Apparatus and method for separating and purifying polynucleotides

ABSTRACT

The disclosure describes an ambient or low pressure device for separating polynucleotide fragments from a mixture of polynucleotide fragments comprises a tube having an upper solution input chamber, a lower eluant receiving chamber, and a fixed unit of separation media supported therein. The separation media has nonpolar separation surfaces which are free from multivalent cations which would react with counterion to form an insoluble polar coating on the surface of the separation media.

RELATIONSHIP TO COPENDING APPLICATIONS

This application is a division of U.S. patent application Ser. No.09/318,407 filed May 25, 1999 (now U.S. Pat No. 6,265,168) which claimspriority from the following U.S. Provisional Patent Applications, eachfiled under 35 U.S.C §111(b):

Serial No. 60/103,313, filed Oct. 6, 1998;

Serial No. 60/117,211 filed Jan. 25, 1999;

Serial No. 60/117,178 filed Jan. 25, 1999;

Serial No. 60/119,945 filed Feb. 12, 1999;

Serial No. 60/123,301 filed Mar. 3, 1999;

Serial No. 60/129,838 filed Apr. 16, 1999; and,

Serial No. 60/130,700 filed Apr. 23, 1999;

and which claims priority from the following commonly assignednon-provisional continuation-in-part U.S. patent applications, eachfiled under 35 U.S.C. §111:

Ser. No. 09/039,061 filed Mar. 13, 1998 (pending);

Ser. No. 09/058,337 filed Apr. 10, 1998 (abandoned);

Ser. No. 09/058,580 filed Apr. 10, 1998 (abandoned);

Ser. No. 09/081,039 filed May 18, 1998 (now U.S. Pat. No. 5,972,222);

Ser. No. 09/129,105 filed Aug. 4, 1998 (now U.S. Pat. No. 6,024,878);

Ser. No. 09/183,047 filed Oct. 30, 1998 (now U.S. Pat. No. 6,066,258);

Ser. No. 09/183,123 filed Oct. 30, 1998 (now U.S. Pat. No. 6,056,877):

Ser. No. 09/183,450 filed Oct. 30, 1998 (now U.S. Pat. No. 5,997,742);

Ser. No. 09/183,573 filed Oct. 30, 1998 (abandoned); and,

Ser. No. 09/311,116 filed May 13, 1999 (now U.S. Pat. No, 6,218,153);

The entire contents of the above-listed patent applications are herebyincorporated by reference.

FIELD OF THE INVENTION

This invention relates to an apparatus and method for separating,isolating, and purifying polynucleotides. In particular, this inventionrelates to methods and devices for separating targeted polynucleotidefragments having a predetermined base-pair length or range of base pairlengths, and for separating and purifying polynucleotides with both highpressure and low pressure devices.

BACKGROUND OF THE INVENTION

A need exists for rapid and efficient procedures for isolating,separating and purifying single-stranded oligonucleotides andsingle-stranded DNA fragments, RNA single-stranded DNA fragments,plasmids and the like. Traditional methods such as ion exchangechromatography, high pressure reverse phase chromatography, gelelectrophoresis, capillary electrophoresis and the like are slow,laborious and inefficient, and they require the services of a highlyskilled chromatographic expert. Furthermore, many methods are incapableof effecting a base-pair length size based separation of these fragmentsand are capable of yielding only minute quantities of separatedmaterials.

Mixtures of single-stranded nucleic acid fragments having different basepair lengths are separated for numerous and diverse reasons. The abilityto detect mutations in single-stranded polynucleotides, and especiallyin DNA fragments which have been amplified by PCR, presents a somewhatdifferent problem since DNA fragments containing mutations are generallythe same length as their corresponding wild type (defined herein below)but differ in base sequence.

DNA separation and mutation detection are of great importance inmedicine, as well as in the physical and social sciences, as well as inforensic investigations. The Human Genome Project is providing anenormous amount of genetic information which is setting new criteria forevaluating the links between mutations and human disorders (Guyer, etal., Proc. Natl. Acad. Sci. USA 92:10841 (1995)). The ultimate source ofdisease, for example, is described by genetic code that differs fromwild type (Cotton, TIG 13:43 (1997)). Understanding the genetic basis ofdisease can be the starting point for a cure. Similarly, determinationof differences in genetic code can provide powerful and perhapsdefinitive insights into the study of evolution and populations (Cooper,et. al., Human Genetics 69:201 (1985)). Understanding these and otherissues related to genetic coding is based on the ability to identifyanomalies, i.e., mutations, in a DNA fragment relative to the wild type.A need exists, therefore, for a methodology which can separate DNAfragments based on size differences as well as separate DNA having thesame length but differing in base pair sequence (mutations from wildtype), in an accurate, reproducible, reliable manner. Ideally, such amethod would be efficient and could be adapted to routine highthroughput sample screening applications.

DNA molecules are polymers comprising sub-units called deoxynucleotides.The four deoxynucleotides found in DNA comprise a common cyclic sugar,deoxyribose, which is covalently bonded to any of the four bases,adenine (a purine), guanine (a purine), cytosine (a pyrimidine), andthymine (a pyrimidine), hereinbelow referred to as A, G, C, and Trespectively. A phosphate group links a 3′-hydroxyl of onedeoxynucleotide with the 5′-hydroxyl of another deoxynucleotide to forma polymeric chain. In single-stranded DNA, two strands are held togetherin a helical structure by hydrogen bonds between, what are called,complimentary bases. The complimentarity of bases is determined by theirchemical structures. In single-stranded DNA, each A pairs with a T andeach G pairs with a C, i.e., a purine pairs with a pyrimidine. Ideally,DNA is replicated in exact copies by DNA polymerases during celldivision in the human body or in other living organisms. DNA strands canalso be replicated in vitro by means of the Polymerase Chain Reaction(PCR).

Sometimes, exact replication fails and an incorrect base pairing occurs,which after further replication of the new strand, results insingle-stranded DNA offspring containing a heritable difference in thebase sequence from that of the parent. Such heritable changes in basepair sequence are called mutations.

In the present invention, single-stranded DNA is referred to as aduplex. When a base sequence of one strand is entirely complimentary toa base sequence of the other strand, the duplex is called a homoduplex.When a duplex contains at least one base pair which is notcomplimentary, the duplex is called a heteroduplex. A heteroduplex isformed during DNA replication when an error is made by a DNA polymeraseenzyme and a non-complimentary base is added to a polynucleotide chainbeing replicated. Further replications of a heteroduplex will, ideally,produce homoduplexes which are heterozygous, i.e., these homoduplexeswill have an altered sequence compared to the original parent DNAstrand. When the parent DNA has a sequence which predominates in anaturally occurring population, it is generally called “wild type”.

Many different types of DNA mutations are known. Examples of DNAmutations include, but are not limited to, “point mutation” or “singlebase pair mutations” wherein an incorrect base pairing occurs. The mostcommon point mutations comprise “transitions” wherein one purine orpyrimidine base is replaced for another and “transversions” wherein apurine is substituted for a pyrimidine (and visa versa). Point mutationsalso comprise mutations wherein a base is added or deleted from a DNAchain. Such “insertions” or “deletions” are also known as “frameshiftmutations”. Although they occur with less frequency than pointmutations, larger mutations affecting multiple base pairs can also occurand may be important. A more detailed discussion of mutations can befound in U.S. Pat. No. 5,459,039 to Modrich (1995), and U.S. Pat. No.5,698,400 to Cotton (1997). These references and the referencescontained therein are incorporated in their entireties herein.

The sequence of base pairs in DNA code for the production of proteins.In particular, a DNA sequence in the exon portion of a DNA chain codesfor the corresponding amino acid sequence in a protein. Therefore, amutation in a DNA sequence may result in an alteration in the amino acidsequence of a protein. Such an alteration in the amino acid sequence maybe completely benign or may inactivate a protein or alter its functionto be life threatening or fatal. On the other hand, mutations in anintron portion of a DNA chain would not be expected to have a biologicaleffect since an intron section does not contain code for proteinproduction. Nevertheless, mutation detection in an intron section may beimportant, for example, in a forensic investigation.

Detection of mutations is, therefore, of great interest and importancein diagnosing diseases, understanding the origins of disease and thedevelopment of potential treatments. Detection of mutations andidentification of similarities or differences in DNA samples is also ofcritical importance in increasing the world food supply by developingdiseases resistant and/or higher yielding crop strains, in forensicscience, in the study of evolution and populations, and in scientificresearch in general (Guyer, et al., Proc. Natl. Acad. Sci. USA 92:10841(1995); Cotton, TIG 13:43 (1997)).

Alterations in a DNA sequence which are benign or have no negativeconsequences are sometimes called “polymorphisms”. In the presentinvention, any alterations in the DNA sequence, whether they havenegative consequences or not, are denoted as “mutations”. It is to beunderstood that the method and system of this invention have thecapability to detect mutations regardless of biological effect or lackthereof. For the sake of simplicity, the term “mutation” will be usedthroughout to mean an alteration in the base sequence of a DNA strandcompared to a reference strand (generally, but not necessarily, wildtype). It is to be understood that in the context of this invention, theterm “mutation” includes the term “polymorphism” or any other similar orequivalent term of art.

A need exists for an accurate and reproducible analytical method formutation detection which is easy to implement. Ideally, the method wouldbe automated and provide high throughput sample screening with a minimumof operator attention, is also highly desirable.

Prior to this invention, size-based analysis of DNA samples relied uponseparation by gel electrophoresis (GEP). Capillary gel electrophoresis(CGE) has also been used to separate and analyze mixtures of DNAfragments having different lengths, e.g., the different lengthsresulting from restriction enzyme cleavage. However, these methodscannot distinguish DNA fragments which differ in base sequence, but havethe same base pair length. Therefore, gel electrophoresis cannot be useddirectly for mutation detection. This is a serious limitation of GEP.

Gel-based analytical methods, such as denaturing gradient gelelectrophoresis (DGGE) and denaturing gradient gel capillaryelectrophoresis (DGGC), can-detect mutations in. heteroduplex DNAstrands under “partially denaturing” conditions. The phenomenon of“partial denaturation” is well known in the art and occurs because aheteroduplex will denature at the site of base pair mismatch at a lowertemperature than is required to denature the remainder of the strand.However, these gel-based techniques are operationally difficult toimplement and require highly skilled personnel. In addition, theanalyses are lengthy and require a great deal of set up time. Adenaturing capillary gel electrophoresis analysis is limited torelatively small fragments. Separation of a 90 base pair fragment takesmore than 30 minutes. A gradient denaturing gel runs overnight andrequires about a day of set up time. Additional deficiencies of gradientgels are the isolation of separated DNA fragments (which requiresspecialized techniques and equipment) and analysis conditions must beexperimentally developed for each fragment (Laboratory Methods for theDetection of Mutations and Polymorphisms, ed. G. R. Taylor, CRC Press,1997). The long analysis time of the gel methodology is furtherexacerbated by the fact that the movement of DNA fragments in a gel isinversely proportional, in a geometric relationship, to their length.Therefore, the analysis time of longer DNA fragments can be often beuntenable.

Recently, an HPLC based ion pairing chromatographic method wasintroduced to effectively separate mixtures of single-strandedpolynucleotides, in general and DNA, in particular, wherein theseparations are based on base pair length. This method is described inthe following references which are incorporated herein in theirentireties: U.S. Pat. No. 5,795,976 (1998) to Oefner; U.S. Pat. No.5,585,236 (1996) to Bonn; Huber, et al., Chromatographia 37:653 (1993);Huber, et al., Anal. Biochem. 212:351 (1993).

As the use and understanding of HPLC developed it became apparent thatwhen HPLC analyses were carried out at a partially denaturingtemperature, i.e., a temperature sufficient to denaturesa heteroduplexat the site of base pair mismatch, homoduplexes could be separated fromheteroduplexes having the same base pair length (Hayward-Lester, et al.,Genome Research 5:494 (1995); Underhill, et al., Proc. Natl. Acad. Sci.USA 93:193 (1996); Doris, et al., DHPLC Workshop, Stanford University,(1997)). These references and the references contained therein areincorporated herein in their entireties. Thus, the use of DenaturingHPLC (DHPLC) was applied to mutation detection (Underhill, et al.,Genome Research 7:996 (1997); Liu, et al., Nucleic Acid Res., 26;1396(1998)).

DHPLC can separate heteroduplexes that differ by as little as one basepair. However, in certain cases, separations of homoduplexes andheteroduplexes are poorly resolved. Artifacts and impurities caninterfere with the interpretation of DHPLC separation chromatograms inthe sense that it may be difficult to distinguish between an artifact orimpurity and a putative mutation (Underhill, et al., Genome Res. 7:996(1997)). The presence of mutations may even be missed entirely (Liu, etal., Nucleic Acid Res. 26:1396 (1998)). The references cited above andthe references contained therein are incorporated in their entiretiesherein.

The accuracy, reproducibility, convenience and speed of DNA fragmentseparations and mutation detection assays based on HPLC have beencompromised in the past because of HPLC system related problems. Thisinvention addresses these problems and applies the term “Matched IonPolynucleotide Chromatography” (MIPC) to the separation method andsystem which is used in connection with the present invention. When usedunder partially denaturing conditions, MIPC is defined herein asDenaturing Matched Ion Polynucleotide Chromatography (DMIPC).

MIPC systems (WAVE® DNA Fragment Analysis System, Transgenomic, Inc. SanJose, Calif.) are equipped with computer controlled ovens which enclosethe columns and column inlet areas. Non-limiting examples of keydistinguishing features of MIPC include the a) use of hardware havingliquid contacting surfaces which do not release multivalent cationstherefrom, b) protection of liquid contacting surfaces from exogenousmultivalent cations by means cartridges containing multivalent cationcapture resins, c) the use of a special washing protocol for MIPCseparation media, d) automated selection of an optimum solvent gradientsolvent gradient for elution of a specific base length DNA fragment, ande) automated determination of the temperature required to effect partialdenaturation of a heteroduplex when MIPC is used under partiallydenaturing conditions (DMIPC) for mutation detection.

The present invention can be used in the separation of RNA or of double-or single-stranded DNA. For purposes of simplifying the description ofthe invention, and not by way of limitation, the separation ofdouble-stranded DNA will be described in the examples herein, it beingunderstood that all polynucleotides are intended to be included withinthe scope of this invention. The invention applies to size-dependentseparations and denaturing separations by MIPC. Both these separationscan include separations of DNA fragments having nonpolar tags.

Important aspects of DNA separation and mutation detection by HPLC andDHPLC which have not been heretofore addressed, comprise a) thetreatment of, and materials comprising chromatography system components,b) the treatment of, and materials comprising separation media, c)solvent pre-selection to minimize methods development time, d) optimumtemperature pre-selection to effect partial denaturation of aheteroduplex during HPLC and e) optimization of DHPLC for automated highthroughput mutation detection screening assays. These factors, whichcomprise MIPC/DMIPC but not HPLC/DHPLC, are essential when usingchromatographic methods in order to achieve unambiguous, accurate,reproducible and high throughput DNA separations and mutation detectionresults. A comprehensive description of MIPC systems and separationmedia, including the critical importance of maintaining an environmentwhich is free of multivalent cations, is presented in U.S. Pat. No.5,772,889 (1998) to Gjerde and U.S. patent application Ser. No.09/129,105 filed Aug. 4, 1998; Ser. No. 09/081,040 filed May 18, 1998(now U.S. Pat. No. 5,997.742); Ser. No. 09/080,547 filed May 18, 1998(now U.S. Pat. No. 6,017,457); Ser. No. 09/058,580 filed Apr. 10, 1998;Ser. No. 09/058,337 filed April 10, 1998; Ser. No. 09/065,913 filed Apr.24, 1998 (now U.S. Pat. No. 5,986,085); Ser. No. 09/039,061 filed Mar.13, 1998; Ser. No. 09/081,039 filed May 18, 1998. These references andthe references contained therein are incorporated in their entiretiesherein.

All of the liquid chromatographic separations discussed herein abovecomprise gradient elution, i.e., they utilize a multi-component mobilephase wherein the concentration of the driving component, usually anorganic solvent, is increased during the course of the chromatography.This approach reduces the time required to complete an analysis.However, the separation of mixture components can be compromised.Efforts have been made to improve the resolving power of MIPC. Theseefforts have centered on improving the gradient process, changing thecolumn particle size, or changing the column length. However, only smallimprovements have been achieved with these efforts. Therefore, thereexists a need improve the separation of poorly resolved or close runningcomponents. Such improvement is especially useful when it is importantto isolate a component in pure form, as for example, for PCRamplification, sequencing, mutation detection, and numerous otherapplications.

Many tasks within molecular biology require prior purification ofnucleic acids. Current strategies involve the use of gel electrophoresisor solid-phase extraction (typically on silica gel or an anion exchangeresin). While these procedures lead to overall improvements in nucleicacid purity relative to original unpurified materials, they suffer fromnegative characteristics. See Hecker, Karl et al, “Optimization ofcloning efficacy by pre-cloning DNA Fragment Analysis”, Biotechnques,26:216-222 February, 1999 which shows the limitations of the prior artseparation methods and the superior purity obtained with the methods ofthis invention. Gel electrophoresis suffers from a lack of automation,incomplete separation of distinct fragments, as well as incompleterecovery of fragments. While solid-phase extraction procedures lendthemselves to automation, they also can suffer from incompleteseparation of distinct fragments and from incomplete recovery offragments.

Many procedures for investigating or evaluating genetic materialsrequire enzymatic cleavage of the materials and isolating a particularDNA fragment or range of DNA fragments from the DNA fragment mixtureproduced by the cleavage. These isolations are particularly important inthe early diagnosis of certain diseases, especially cancer. In the caseof cancer and other diseases of genetic origin, early detection oftendepends on the availability of an appropriate analytical method whichcan accurately and reliably detect a mutation in DNA samples.

As described in copending Sklar et al U.S. patent application Ser. No.09/311,116 filed May 13, 1999, this problem is exacerbated by the factthat such samples may contain a very small population of cellscontaining mutant DNA in the presence of a very large predominantlynormal cell population containing, for example, wild type DNA. Beforethe development of this invention, any separation techniques which weretheoretically capable of detecting mutant DNA in the presence of wildtype would fail because the concentration of mutant DNA was simply toolow to be detected relative to wild type. That is to say, theconcentration of mutant DNA may be too low to detect in absolute terms.Alternatively, the concentration of mutant DNA may be sufficient todetect, but will be completely obscured because of the very largerelative amount of wild type in the sample.

Increasing the amount of mutant DNA by PCR amplification of the samplewould not solve the problem described above. The mutant and wild typeDNA in the sample are very similar. In fact, their sequence may differby only a single base pair. Therefore, the primers used to amplify themutant DNA would also amplify the wild type since both are present inthe sample. As a result, the relative amounts of mutant and wild typeDNA would not change.

For example as described in the copending Sklar et al application(supra), following radiation or chemotherapy, cancer patients aremonitored for the presence of residual cancer cells to determine whetherthe patients are in remission. The effectiveness of these treatments canbe monitored if small levels of residual cancer cells could be detectedin a predominantly large wild type population. Traditionally, theremission status is assessed by a pathologist who conducts histologicalexamination of tissues samples. However, these visual methods arelargely qualitative, time-consuming, and costly. At best, thesensitivity of these methods permits detection of about 1 cancerous cellin 100 cells.

Analysis of DNA samples has historically been done using gelelectrophoresis. Capillary electrophoresis has also been used toseparate and analyze mixtures of DNA. However, these methods cannotdistinguish point mutations from homoduplexes having the same base pairlength.

In addition to the deficiencies of denaturing gel methods mentionedabove, these techniques are not always reproducible or accurate sincethe preparation of a gel slab and running an analysis can be highlyvariable from one operator to another. As a result, the mobility of aDNA fragment is different on different gel slabs and even in one lane,compared to another on the same gel slab. The problems and deficienciesof gel-based DNA separation methods are well known in the art and aredescribed in the published literature, e.g., G. R. Taylor, editor,LABORATORY METHODS FOR THE DETECTION OF MUTATIONS AND POLYMORPHISMS, CRCPress (1997).

SUMMARY AND OBJECTS OF THE INVENTION

It is an object of this invention to provide a method and apparatus forrapid and efficient base-pair length size separation of double-strandedDNA (dsDNA) and for effecting size-based separation of single-strandedoligonucleotides and single-stranded DNA fragments, RNA, plasmids andthe like.

It is a further object of this invention to provide a simple andinexpensive method and apparatus for size-based separation of thesefragments, which can be easily and reproducibly operated by a trainedtechnician.

It is a still further object of this invention to provide a method anddevices for low pressure separation of these fragments.

It is another object of this present invention to provide a sensitiveand reproducible method enabling the isolation of small amounts of anytarget DNA in a mixture of DNA fragments.

It is a still further object of this invention to provide a method andsystem which is suitable for isolating a mutant DNA in the presence of arelatively large amount of wild type DNA, wherein such mutations wouldotherwise go undetected.

It is a principal object of this invention to provide an analyticalmethod which is reproducible, reliable, inexpensive, can be automatedand can be used for high throughput sample screening.

In summary, a method of this invention for isolating targeted DNAfragments having a predetermined base-pair length from a mixture of DNAfragments comprises the following steps. The mixture of DNA fragments isapplied to a separation column containing separation media having anonpolar, nonporous surface, the mixture of DNA fragments being in afirst solution containing counterion and a DNA binding concentration ofdriving solvent. Targeted DNA fragments are removed from the media bycontacting the media with a second solution containing counterion and aconcentration of driving solvent which has been predetermined to removeDNA fragments having the targeted fragments from the separation mediainto a distinct segment of eluant.

Optionally, the targeted DNA fragments are recovered. The recoveredtargeted DNA fragments can be amplified and cloned.

The method can be applied to determine the presence or absence of DNAfragments having a specific base-pair length in the sample mixture. Themethod can be applied when the DNA fragments having said base pairlength are present in the sample mixture in a concentration which is toolow to be detected in the analysis, and the amplified product isanalyzed to verify the presence of DNA fragments having said base pairlength in the sample mixture.

Another method of this invention is a procedure separating by DMIPCtarget homoduplex and/or heteroduplex DNA fragments from a mixture ofhomoduplex and heteroduplex DNA fragments having the same base-pairlength, the heteroduplex fragments having at least one mismatch site.The method comprises the following steps: The mixture of homoduplex andheteroduplex DNA fragments is applied to a separation column containingseparation media having a nonpolar, nonporous surface, the mixture ofhomoduplex and heteroduplex DNA fragments being applied in a firstsolution containing a counterion and a DNA binding concentration ofdriving solvent. While maintaining the media and the solution at atemperature which will locally denature the heteroduplex fragments atthe mismatch site thereof, separating desired homoduplex and/orheteroduplex DNA fragments from the separation media by contacting theseparation media with a second solution containing a counterion and aconcentration of driving solvent which has been predetermined toseparate the target homoduplex and/or heteroduplex DNA fragments fromthe separation media in separate fractions. When the desired fragmentsare a homoduplex fraction, the concentration of driving solvent isselected to remove one or both of the homoduplex fractions as separatefractions from homoduplex fractions. Alternatively, when the desiredfragments are a heteroduplex fraction, the concentration of drivingsolvent is selected to remove one or both of the heteroduplex fractionsas separate fractions from homoduplex fractions.

The heteroduplex fraction can be collected and amplified. The amounts ofthe heteroduplex fragments to homoduplex fragments is less than 1:1. Themethod can be used when the amount of heteroduplex in the separateheteroduplex fraction is below the level of detection.

The retention time used in the separation of the homoduplex orheteroduplex fraction can be previously determined from a referencestandard. The reference standard can be obtained by separating astandard mixture of homoduplex and heteroduplex, having the same basepair sequence as the sample, by Matched Ion PolynucleotideChromatography.

This method can be used to determine the presence or absence of ahomoduplex or a heteroduplex faction in a mixture of homoduplex andheteroduplex DNA fragments having the same base-pair length, theheteroduplex fragments having at least one mismatch site. The recoveredfragments can be cloned. The method is applicable when the DNA samplecontains a large background of wild type and when the mutant DNA isbelow the limit of detection, when the DNA sequences of the wild typeDNA and the mutant DNA are known, and when the mutant DNA differs fromwild type DNA by at least one base pair.

In summary, an ambient or low pressure device of this invention forseparating polynucleotide fragments from a mixture of polynucleotidefragments comprises a tube having an upper solution input chamber, alower eluant receiving chamber, and a fixed unit of separation mediasupported therein. The separation media has nonpolar separation surfaceswhich are free from multivalent cations which would react withcounterion to form an insoluble polar coating on the surface of theseparation media.

The separation media can be beads, capillary channels or monolithstructures. The fixed unit of separation media can comprise a fixed bedof separation media particles, and the separation media particles can beorganic polymer or inorganic particles having a nonpolar surface.

In one embodiment, the lower chamber is closed. In other embodiments,the lower chamber has an open bottom portion. The tube can be combinedwith a eluant container shaped to receive the lower chamber. The eluantchamber can be a centrifuge vial.

In one embodiment, the tube is a member of an array of tubes and theeluant container is a member of an array of eluant containers, and thearray of tubes and array of containers have matching configurations.

An ambient or lower pressure separation system of this inventioncomprises a combination of multicavity separation plate having outersealing edges, a multiwell collection plate and a vacuum system. Thevacuum system has a separation plate sealing means forming a sealedengagement with the outer sealing edges of the multicavity separationplate and a vacuum cavity receiving the multiwell collection plate. Themulticavity separation plate includes an array of tubes, each tubehaving an upper solution input chamber, a lower eluant receiving chamberwith a bottom opening therein, and a fixed unit of separation mediasupported therein. The separation media can have nonpolar separationsurfaces which are free from multivalent cations which would react withcounterion to form an insoluble polar coating on the surface of theseparation media. The multiwell collection plate has collection wellswhich are positioned to receive liquid from the bottom opening of thelower eluant receiving chamber. The separation media can be beads,capillary channels or monolith structures. The fixed unit of separationmedia comprises a fixed bed of separation media particles. Theseparation media particles can be selected from the group consisting oforganic polymer and inorganic particles having a nonpolar surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a high pressure system forperforming the MIPC method of this invention, with a proportioning valvesystem for effecting gradients of solvent concentrations in theseparation.

FIG. 2 is a partial schematic representation corresponding to therepresentation of FIG. 1, but with a proportioning pump system foreffecting gradients of solvent concentrations in the separation systemwith proportioning valve

FIG. 3 is a cross-sectional view of a high pressure separation columnsuitable for use in the apparatus of FIGS. 1 and 2.

FIG. 4 is a schematic representation of hybridization of wild type DNAstrand with homozygous mutant strand showing the production of twohomoduplexes and two heteroduplexes.

FIG. 5 is a DMIPC chromatogram showing the separation of a standardmixture of FIG. 4.

FIG. 6 shows DMIPC chromatograms demonstrating mutation detection usingthe procedures of blind target zone elution.

FIG. 7 is a cross-sectional representation of a spin vial system for lowpressure separations according to this invention.

FIG. 8 is a multiwell plate separation system of this invention incombination with a vacuum attachment.

FIG. 9 is the top view of a multiwell plate of FIG. 8.

FIG. 10 is a cross-sectional view of the separation tray of FIG. 8 takenalong the line A—A.

FIG. 11 is an enlarged view of a single separation cell of the multiwellplate of FIG. 10.

FIG. 12 is a chromatogram of the unseparated mixture of DNA fragments inthe procedure of Example 1.

FIG. 13 is a chromatogram of the separated 80 bp fragment obtained inthe procedure of Example 1.

FIG. 14 is a chromatogram of the separated 102 bp fragment obtained inthe procedure of Example 1.

FIG. 15 is a chromatogram of the separated 174 bp fragment obtained inthe procedure of Example 1.

FIG. 16 is a chromatogram of the separated 257 bp fragment obtained inthe procedure of Example 1.

FIG. 17 is a chromatogram of the separated 267 bp fragment obtained inthe procedure of Example 1.

FIG. 18 is a chromatogram of the separated 298 bp fragment obtained inthe procedure of Example 1.

FIG. 19 is a chromatogram of the separated 434 bp fragment obtained inthe procedure of Example 1.

FIG. 20 is a chromatogram of the separated 458 bp fragment obtained inthe procedure of Example 1.

FIG. 21 is a chromatogram of the separated 587 bp fragment obtained inthe procedure of Example 1.

FIG. 22 is a chromatogram of a pUC 18 Msp I standard mixture of dsDNAfragments used in Example 3.

FIG. 23 is a chromatogram of the low molecular weight and smallbase-pair length fraction eluant obtained in Example 3.

FIG. 24 is a chromatogram of the high base-pair length fraction eluantobtained in Example 3, demonstrating the efficacy of the spin columndevice for purifying high base-pair length components of a mixture ofDNA fragments.

FIG. 25 is a chromatogram of a pBR322 standard mixture of dsDNAfragments used in Example 4.

FIG. 26 is a chromatogram of the low molecular weight and smallbase-pair length fraction eluant obtained in Example 4.

FIG. 27 is a chromatogram of the high base-pair length fraction eluantobtained in Example 4.

FIG. 28 is a chromatogram obtained in the procedure of Example 5.

FIG. 29 is a chromatogram obtained in the procedure of Example 5.

FIG. 30 is a chromatogram obtained in the procedure of Example 6.

FIG. 31 is a chromatogram of the separated hybridized dsDNA mixtureobtained by elution with an isocratic mobile phase.

FIG. 32 illustrates the hysteresis effect obtained in kineticseparation.

DETAILED DESCRIPTION OF THE INVENTION

Although the method of the invention relates to polynucleotides ingeneral, the discussion to follow will reference double-stranded DNA(dsDNA) for the sake of simplicity only and not by way of limitation.

The term “Matched Ion Polynucleotide Chromatography” as used herein isdefined as a process for separating single and double-strandedpolynucleotides using nonpolar separation media, wherein the processuses a counterion agent, and an organic solvent to release thepolynucleotides from the separation media. MIPC separations are completein less than 10 minutes, and frequently in less than 5 minutes. MIPCsystems (WAVE® DNA Fragment Analysis System, Transgenomic, Inc. SanJose, Calif.) are equipped with computer controlled ovens which enclosethe columns and sample introduction areas.

A “gradient”, as defined herein, is a chromatographic mobile phasedefined by an initial time point having an initial solvent compositionand a final time point having a final solvent composition which isdifferent from the initial solvent concentration, and the gradientcomposition continually changes (e.g. usually increases) from theinitial solvent composition to the final solvent composition over thetime interval between the initial and final time points. A gradientmobile phase is used to elute fragments from a chromatography column forthe purpose of separating the components in a mixture.

The term “isocratic” is defined herein to denote a chromatographicmobile phase whose composition remains essentially constant for any partof or all of the duration of the chromatographic separation process. Theterm “isocratic” is intended to include a process wherein a singlesolvent concentration is maintained throughout the separation, a processwhere the solvent concentration is stepped from one constantconcentration to one or more constant concentrations in a sequence ofsteps, or a process with a gradient separation and with one or moreportions conducted under constant solvent concentration conditions. Anisocratic mobile phase is used to elute fragments from a chromatographycolumn for the purpose of separating a mixture thereon, into itscomponents (Remington: The Science and Practice of Pharmacy, 19^(th)Ed., A. Gennaro, Ed. p. 537 (1995)). This reference is incorporated inits entirety herein. The isocratic separation solvent concentrationshould be maintained within ±1% of the selected isocratic solventconcentration and is preferably maintained within ±0.5% of the selectedisocratic solvent concentration. Optimally, the isocratic solventconcentration is maintained with ±0.1% of the selected isocratic solventconcentration.

The term “partially denaturing” means the separation of a mismatchedbase pair (caused by temperature, pH, solvent, or other factors) in aDNA double strand while the remainder of the double strand remainsintact.

There is generally a wide range of interactions with the column.Typically, fragments of many various sizes are analyzed. For example,separation of fragments of 80 to 600 base pairs on MIPC columns requireacetonitrile concentrations of about 8.75% to 16.25%, respectively.Longer fragments require further increases in acetonitrileconcentrations. Therefore, to perform separation of an entire range offragment sizes, a gradient process is necessary. Otherwise, it is notpossible to separate this range of sizes.

The apparatus of this invention provides a novel and unique method forseparating and purifying double-stranded oligonucleotides andsingle-stranded DNA fragments, RNA, double-stranded DNA fragments,plasmids and the like. The device simplifies the separation procedureand applies a unique size-based separation process based on our MatchedIon Pair Chromatography (MIPC), also denoted herein by the term DNAChromatography. This process exploits the binding characteristics withnonpolar surfaces of separation media in the presence of counterion andmaterials to be separated. Materials in aqueous solutions of thecounterion and low stripping solvent concentrations bind to the nonpolarsurfaces, and the materials are subsequently released from the surfaceby application of a stripping solvent concentration which removes orstrips materials from the separation media surface, the size of thematerials being stripped being a function of the stripping solventconcentration. Larger-sized materials require application of a greaterstripping solvent concentration to effect their release. The ratio offragment size desorbed from the media to the concentration of strippingsolvent can be calibrated and is so reproducible that it can becalculated with high accuracy. The process can be applied with anysystem which can retain the separation media and provides means torapidly pass liquids through the separation media.

FIG. 1 is a schematic representation of a high-pressure system forperforming the MIPC method of this invention, with a proportioning valvesystem for effecting gradients of solvent concentrations in theseparation. Chromatographic solutions such as solvents, counterions, andother solutions to be mixed with the solvents are maintained in solventcontainer 2, carrier liquid container 4, and auxiliary liquid (e.g., acosolvent) container 6 having respective solvent transport tubing 8,carrier transport tubing 10 and auxiliary liquid transport tubing 12communicating therewith and leading to degasser 14.

Column cleaning solution is contained in cleaning solution container 16which likewise has a cleaning solution transport conduit 18communicating therewith leading to the degasser 14. In this embodiment,the cleaning solution can flow by gravity pressure if the container 16is elevated above the degasser and injection valve 54. Alternatively, apump as shown in FIG. 2 can be provided to achieve cleaning solutionflow.

Degassed solvent conduit 20, degassed carrier liquid conduit 22, anddegassed auxiliary liquid conduit 24 leading from the degasser 16communicate with respective solvent proportioning valve 26, carrierliquid proportioning valve 28, and auxiliary liquid proportioning valve30. The settings for these proportioning valves are set and changed byvalve operators such as stepper motors associated therewith, and thesevalve operators respond to establish a desired set of settings inresponse to commands from the valve operator control module described ingreater detail hereinafter. The settings for these proportioning valvescontrol the ratio of liquids (co-solvents, driving solvents, etc.)through the injector valve and the separation column. Conduits 32, 34,and 36 lead from respective proportioning valves 26, 28 and 30 to theintake of the pump 38. The degasser 14 removes dissolved gases from theliquids. Removal of dissolved oxygen is particularly important becauseits presence increases the risk of oxidizing ferrous or other oxidizablemetals in the system components and thus introducing the correspondingcations into the liquid.

The cleaning solution transport conduit 31 leads to a cleaning solutionvalve 40. An optional cleaning solution conduit 42 leads from the valve40 and communicates with the inlet of the pump 38.

The openings of valves 26, 28 and 30 accurately set the relative ratiosof the solvent or solvents to carrier liquid, a most important part ofthis system because the size-based DNA separation by MIPC is a functionof solvent concentration. As will be described with regard to thevarious DNA fragment separation processes, the slope of the solventgradient as a function of time is changed during the separation process,and the most critical phase may require a very precise gradient, or forsome processes, a highly precise isocratic (constant solventconcentration) composition. The settings of the valves 26, 28 and 30 areestablished by conventional valve actuators which can be remotely set bysignals to a conventional valve control device. As will be described ingreater detail hereinafter, the control system of this inventionprovides computer controlled instructions which establish the settingsof valves 26, 28 and 30 to precise flow values at appropriate timesduring the operation of the system.

In a similar manner, the control system of this invention providescomputer controlled instructions to establish the operational parametersof the pump 38, such as the off/on status-of the pump and the pressureor flow rate settings of the pump.

Pump outflow conduit 44 communicates with the in-line mixer 46,directing the liquid flow through the mixer 46 for thorough mixing ofthe components. Mixed liquid outflow conduit 48 communicates with guardcolumn 50 to treat the mixed liquid to remove multivalent metal cationsand other contaminants which would interfere with the separation of DNAfragments. Guard column 50 can contain a cation exchange resin in sodiumor hydrogen form for removal of multivalent metal cations byconventional ion exchange. Conduit 52 communicates with the outlet ofthe guard column and an inlet port of a cleaning solution injector valve54. Waste supply conduit 56 connects valve 40 with the cleaning solutioninjector valve 54, and waste outlet conduit 58 leads to waste. Conduit60 leads from the waste solution injector valve 54 to the sampleinjection valve 62.

Sample aliquot selector 64 communicates with injector valve 62 throughsample conduit 66. Waste conduit 68 leads from the injector valve andremoves waste liquids.

In the injector valve 62, the sample is introduced into a stream ofsolvent and carrier liquid passing through the valve from conduit 60.Sample conduit 70 communicates with an outlet port of injector valve 62and with the column prefilter 74 in the air bath oven 72. The capillarytubing coil 76 communicates with the prefilter 74 and the inlet ofseparation column 78. The extended length of the capillary coil 76allows ample heat to pass from the heated oven air into the liquidpassing through the coil, bringing the liquid within ±0.05° C. of aselected temperature. The oven 72 establishes this temperatureuniformity in the prefilter 74, coil 76, and separation column 78.

The separation column 78 is packed by conventional column constructionwith beads having a unique separation surface which effects a size-basedseparation of DNA fragments in the presence of a matched counterion bythe MICP process. The separation process and details about the beads aredescribed in detail hereinafter. A stream (eluant) containing base pairlength size-separated DNA fragments passes from the separation column 78through eluant conduit 80.

Analyzer 84 communicates with conduit 80. The analyzer cell 84 can be aconventional UV absorbance measurement device which measures the UVabsorbance level of the native DNA fragment structures in the liquid.The absorption level is a function of the concentration of the DNAfragments in the liquid being tested.

Alternatively, if the DNA is labeled with a fluorescent marker, theanalyzer continuously measures the level of the fluorescent marker inthe liquid by detecting the emission level at the frequency mostappropriate for the marker. Similarly, if the DNA is labeled with a masstag, the detector can be a mass spectrophotometer. It will be readilyapparent that any analyzing system capable of continuously measuring acharacteristic of the liquid which is a function of the concentration ofthe DNA fragments therein is suitable and intended to be within thescope of this invention

The eluant passes from the analyzer 84 to the fragment collector 88. Inthe fragment collector 88, selected portions of the eluant containing aseparated DNA fraction are collected in a vials for later processing oranalysis. Uncollected fractions are removed through waste conduit 90.

The DNA separation process is impaired by the presence of multivalentcations. In the above description, the liquid flow system is describedas a series of conduits. The conduits are capillary tubing selected toavoid introduction of multivalent cations into the liquids. Thepreferred capillary tubing materials are titanium and PEEK. For similarreasons, the other components of the system are preferably made oftitanium or PEEK or have the surfaces exposed to the liquid coated withPEEK to protect them from oxidation and prevent the introduction ofmultivalent cations into the liquid. Moreover, titanium and PEEK parts,tubing and surfaces can be additionally treated to remove contaminants.

Stainless steel can also be used provided it has been treated to removeall oxidized surface materials and the solutions contacting thestainless steel surfaces are free of dissolved oxygen.

FIG. 2 is a partial schematic representation corresponding to therepresentation of FIG. 1, but with a proportioning pump system foreffecting gradients of solvent concentrations in the separation. Thissystem relies on proportioning pumps to control the ratio of solvents tocarrier liquids. The inlets of proportioning pumps 92, 94 and 96 by wayof their respective supply conduits 98, 100, and 102 communicate withthe degasser 14, and by way of their respective outlet conduits 104, 106and 108 communicate with the inline mixer 46. The operational speeds forthese proportioning pumps are calibrated to flow rates therethrough andare controlled by a proportioning pump control module described ingreater detail hereinafter. The settings for these proportioning valvescontrol the liquid flow speed and the ratio of liquids (co-solvents,driving solvents, etc.) through the injector valve and the separationcolumn.

A pump 110 can supply cleaning solution to the system through optionalconduit 112. An optional conduit 113 can lead from conduit 112 andcommunicate with the in-line mixer 46.

FIG. 3 is a partially exploded representation of the physical structureof a representative separation column. The column comprises a columntube 120 with external threads on both ends. The tube is filled withseparation media 122. A frit 124 is held against the upper surface ofthe separation media by the frit plug 126. An internally threadedcoupling 128 is secured to the end of the tube 120 and receives the frit124 and frit plug 126. The internally threaded coupling 128 receives theexternally threaded coupling 130 in a threaded engagement. Theexternally threaded coupling 130 has an internally threaded end receptor132 for receiving a capillary tubing end coupler (not shown).

The separation media 122 are organic polymer materials or inorganicmaterials having the requisite structure and nonpolar surfaces asdescribed in greater detail hereinafter.

The methods of this invention for isolating targeted DNA fragmentshaving a predetermined base-pair length from a mixture of DNA fragmentscomprise the following steps. A mixture of DNA fragments is applied to aseparation column containing separation media having a nonpolar,nonporous surface, the mixture of DNA fragments being in a firstsolution containing counterion and a DNA binding concentration ofdriving solvent. The targeted DNA fragments are then removed from themedia by contacting the media with a second solution containingcounterion and a concentration of driving solvent which has beenpredetermined to remove DNA fragments having the targeted fragments fromthe separation media into a distinct segment of eluant.

The present invention relates, therefore, to the isolation of DNAfragments having a predetermined base pair (bp) length from a mixture offragments. It also relates to the isolation in a mixture of DNAfragments having an identical bp length, heteroduplex fragments fromhomoduplex fragments, heteroduplex fragments from one another andhomoduplex fragments from one another. For example, the technology ofthis invention can be applied to the Sklar et al process (supra) toachieve an unambiguous detection and identification of very smallamounts of heteroduplex fragments containing mutant DNA in the presenceof a relatively very large amount of known wild type using ourDenaturing Matched Ion Polynucleotide Chromatography (DMIPC), a methodanalogous to Matched Ion Polynucleotide Chromatography (MIPC).

MIPC separates DNA fragments based on their base pair length (U.S. Pat.No. 5,585,236 to Bonn (1996); Huber, et al., Chromatographia 37:653(1993); Huber, et al., Anal. Biochem. 212:351 (1993)). These referencesand the references contained therein are incorporated herein in theirentireties. When MIPC analyses are performed at partially denaturingtemperature, the process is called DMIPC. These separation methodsobviate the deficiencies of gel-based methods and make possible thecollection and identification of mutant DNA fragments whoseconcentration relative to wild type is small, and may be below thedetection limits of a detector. Alternatively, MIPC and DMIPC makepossible the collection and identification of mutant fragments whichwould be obscured by a relatively large amount of wild type in a sample,as exemplified in the Sklar et al process (supra). This method will bediscussed in detail herein below.

MIPC uses unique nonpolar separation media which comprises organicpolymers, silica media having a nonpolar surface comprising coated orcovalently bound organic polymers or covalently bound alkyl and/or arylgroups, and continuous nonpolar separation media, i.e., beads, monolithor rod columns, capillary tubing or blocks containing capillary sizedpassageways or channels, or other nonpolar surfaces of material such asorganic polymer or nonpolar surfaced silica gel. The separation mediaused in MIPC can be porous or nonporous. A detailed description of theMIPC separation process, MIPC separation media, and MIPC systems isfound in U.S. Pat. No. 5,772,889 (1998) to Gjerde and in co-pending U.S.patent applications Ser. No. 09/058,580 filed Mar. 10, 1998; Ser. No.09/058,337 filed Mar. 10, 1998; Ser. No. 09/081,040 filed May 18, 1998;Ser. No. 09/080,547 filed May 18, 1998; Ser. No. 09/169,440 filed Oct.,9, 1988 (pending); Ser. No. 09/183,123 filed Oct. 20, 1998; and Ser. No.09/183,450 filed Oct. 20, 1998. MIPC systems and separation media arecommercially available (WAVE™ DNA separation system, WAVEMAKER™ DNAseparation system software and DNASEP® DNA separation columns fromTransgenomic, Inc. San Jose, Calif.). The entire MIPC analysis can beautomated by means of a desk top computer and a sample auto-injector.Analytical data for each sample can be analyzed in real time, orcollected and stored in a computer memory device for analysis at a latertime.

The use of MIPC at partially denaturing temperature, i.e., DMIPC, todetect mutations has been described in a co-pending U.S. patentapplication Ser. No. 09/129,105 filed Aug. 4, 1998. This application andthe references contained therein are incorporated herein in theirentireties.

The MIPC method is highly reproducible. Therefore, columns do not haveto be calibrated from sample to sample or from day to day. A DNAfragment of a particular base pair length will elute from an MIPC columnat a specific retention time which is reliably reproducible. Thischaracteristic, coupled with the automation, sample collection, andrapid sample analysis capabilities of MIPC make this method uniquelysuited for detection of minute quantities of mutations in the presenceof a large background of wild type.

The MIPC method of this invention has been found to involve a uniquerelationship between the concentration of the organic (driving) solventin the mobile phase and the hydrophobic stationary phase. The strongaffinity of the polynucleotide fragments for the stationary phasedominates the interactions until the solvent concentration reaches acritical range for each fragment size. Within the critical range, thesolvent competes with the stationary phase, and the fragment is releasedor desorbed and quickly reaches the velocity of the eluant. Forseparation processes using a solvent gradient, the fraction released isa simple direct function of the solvent concentration, a binding andrelease relationship which is not column length dependent.

If the solvent concentration is maintained at a constant level withinthe critical range for a fragment size (isocratic separation), thecompetitive phase is attenuated, and the separation of the fractions inthe eluant shows some enhancement with increased column length. If thesolvent concentration changes in a very slight gradient through thecritical range for a fragment size, the rate of full release of thefragment into the eluant is increased, and the competitive phase isquickly diminished.

In other words, when the solvent gradient favors the quick release ofthe fragments, the column length becomes insignificant because thecomplete separation process occurs within a very short distance at thetop of the column. As the slope of the solvent gradient is reduced andnears an isocratic process, the competition with the stationary phaseextends over an increasing length of the column, delaying full releaseof the fraction into the eluant.

With the MIPC separation process, in contrast, the DNA is applied to thenonpolar, nonporous surfaces of the separation media in a dilutesolution of driving solvent, cosolvent, and counterion. The DNA binds atthe top of a column. We have discovered that the base pair lengthdetermined separation of the DNA from the separation media is entirely afunction of driving solvent concentration in the cosolvent. Controllingthe driving solvent concentration as function of time isolates a targetDNA fragment from the column as a function of time in an entirelypredictable manner.

The method can be used to collect and amplify the target DNA fragments.The procedure can be used as an assay to determine whether or not thetarget DNA fragments are present in the sample. So the method can beapplied when no DNA fragments having the base pair length of the targetDNA are present and when DNA fragments having the base pair length ofthe target DNA are present.

Isocratic elution provides far better separation of component fragmentshaving small differences in lengths than could be achieved with agradient elution. Therefore, in one embodiment, the present inventionprovides a method for enhancing the separation of dsDNA fragments on anMIPC column by using an isocratic mobile phase to elute fragments fromthe column.

Separations of dsDNA fragments by MIPC under isocratic elutionconditions are extremely sensitive to solvent composition. Small changesin the composition of solvent mixtures contained in solvent reservoirscan occur as a result of evaporation. Such changes can result indifferences in the retention times of fragments from identical samples.To prevent changes in retention time resulting from changing solventcomposition it is important to keep solvent reservoir bottles tightlyclosed with caps, PARAFILM, aluminum foil, or similar materials. Inaddition, when helium sparging is used to de-gas solvents, the flow ofgas should be reduced to a minimum in order to minimize the evaporationof solvent. The use of an in-line degasser is a preferred method used tominimize solvent evaporation and maintain constant solvent composition.An example of a suitable degasser is DEGASET, Model 6324 (MetaChemTechnologies, Torrance, Calif.).

There are several types of counterions suitable for use with MIPC. Theseinclude a mono-, di-, or trialkylamine that can be protonated to form apositive counter charge or a quaternary alkyl substituted amine thatalready contains a positive counter charge. The alkyl substitutions maybe uniform (for example, triethylammonium acetate or tetrapropylammoniumacetate) or mixed (for example, propyldiethylammonium acetate). The sizeof the alkyl group may be small (methyl) or large (up to 30 carbons)especially if only one of the substituted alkyl groups is large and theothers are small. For example octyldimethylammonium acetate is asuitable counterion agent. Preferred counterion agents are thosecontaining alkyl groups from the ethyl, propyl or butyl size range.

The mobile phase preferably contains a counterion agent. Typicalcounterion agents include trialkylammonium salts of organic or inorganicacids, such as lower alkyl primary, secondary, and lower tertiaryamines, lower trialkyammonium salts and lower quaternary alkylammoniumsalts. Examples of counterion agents include octylammonium acetate,octadimethylammonium acetate, decylammonium acetate, octadecylammoniumacetate, pyridiniumammonium acetate, cyclohexylammonium acetate,diethylammonium acetate, propylethylammonium acetate,propyldiethylammonium acetate, butylethylammonium acetate,methylhexylammonium acetate, tetramethylammonium acetate,tetraethylammonium acetate, tetrapropylammonium acetate,tetrabutylammonium acetate, dimethydiethylammonium acetate,triethylammonium acetate, tripropylammonium acetate, tributylammoniumacetate. Although the anion in the above examples is acetate, otheranions may also be used, including carbonate, phosphate, sulfate,nitrate, propionate, formate, chloride, and bromide, or any combinationof cation and anion. These and other agents are described by Gjerde, etal. in Ion Chromatography, 2nd Ed., Dr. Alfred Hüthig Verlag Heidelberg(1987). Counterion agents that are volatile are preferred for use in themethod of the invention, with triethylammonium acetate (TEAA) andtriethylammonium hexafluoroisopropyl alcohol being most preferred.

To achieve optimum peak resolution during the separation of DNA by MIPC,the method is preferably performed at a temperature within the range of20° C. to 90° C.; more preferably, 30° C. to 80° C; most preferably, 50°C. to 75° C. The flow rate is selected to yield a back pressure notexceeding 5000 psi. In general, separation of single-stranded fragmentsshould be performed at higher temperatures.

The temperature at which the separation is performed affects the choiceof organic solvents used in the separation. One reason is that thesolvents affect the temperature at which a double-stranded DNA will meltto form two single strands or a partially melted complex of double andsingle-stranded DNA. Some solvents can stabilize the melted structurebetter than other solvents. The other reason a solvent is important isbecause it affects the distribution of the DNA between the mobile phaseand the stationary phase. Acetonitrile and 1-propanol are preferredsolvents in these cases. Finally, the toxicity (and cost) of the solventcan be important. In this case, methanol is preferred over acetonitrileand 1-propanol is preferred over methanol.

When the separation is performed at a temperature within the aboverange, an organic solvent that is water soluble is preferably used, forexample, alcohols, nitriles, dimethylformamide (DMF), tetrahydrofuran(THF), esters, and ethers. Water soluble solvents are defined as thosewhich exist as a single phase with aqueous systems under all conditionsof operation of the present invention. Solvents which are particularlypreferred for use in the method of this invention include methanol,ethanol, 2-propanol, 1-propanol, tetrahydrofuran (THF), andacetonitrile, with acetonitrile being most preferred overall.

Size based separations using an isocratic mobile phase are bestperformed below a temperature which will denature a dsDNA. Thistemperature range comprises 25° C. to about 55° C., with about 50° C.being most preferred.

One aspect of this invention is a method for determining the presence orabsence of DNA fragments having a specific base-pair length in a samplemixture. The method comprises the steps of applying the sample mixtureto a separation column containing separation media having a nonpolar,nonporous surface, the sample mixture being in a first solvent mixturecontaining a counterion and a DNA binding concentration of drivingsolvent in a cosolvent. Then any DNA in the sample having said specificbase pair length is removed by contacting the separating media with asecond solvent solution containing a counterion and a concentration ofdriving solvent which has been predetermined to remove DNA fragmentshaving said base pair from the separation media. The separated fractioncan be amplified by PCR, for example, to increase the amount of theputative fraction or confirm its absence from the sample.

This invention applies this discovery to purify and isolate mutantfragments by “blind collection”. The term “blind collection” is definedherein to mean the collection of mobile phase flowing through an MIPCcolumn over a specific time interval subsequent to application of a DNAsample to the column. More specifically, “blind collection” refers tocollecting mobile phase during the retention time interval correspondingto a previously determined retention time interval of a DNA fragmentstandard. Since the relationship between MIPC retention time and basepair length is highly reproducible, it is not necessary to detect adesired fragment with a detector in order to know when to collect thefragment. Column mobile phase is simply collected at the predeterminedand expected retention time of a desired fragment.

Use of a specific concentration of organic component in the isocraticmobile phase results in enhanced separation dsDNA within a relativelynarrow range of base pair lengths, referred to herein as a “targetrange” of base pair lengths. Therefore, in another embodiment, a mixtureof dsDNA can be separated using a gradient mobile phase to-separatefragments within a selected base pair range. Chromatographic fractionscontaining fragments within the target range can be isolated andre-chromatographed using an isocratic mobile phase. The enhancedseparation afforded by the use of an isocratic mobile phase makespossible the isolation of one or more pure fragments within the targetrange. Such pure fragments can then be amplified using PCR to providerelatively large quantities of high purity product. High purity dsDNAfragments have many uses, e.g., sequencing, forensic investigations,cloning and sample preparation prior to DNA analysis.

In another embodiment, the present invention can be used to detect thepresence or absence of mutations. In this embodiment, a sample dsDNA ishybridized with wild type. Hybridization, a standard process in thebiotechnology art, is effected by heating a solution of sample and wildtype DNA to about 90° C. for about 5 minutes, then slowly cooling thesolution to ambient temperature over 45 to 60 minutes. During theheating period, the dsDNA strands denature. Upon slow cooling, theyrecombine in a statistical fashion. Therefore, if the sample contains amutation, the hybridized product will contain a mixture of twohomoduplexes and two heteroduplexes. A schematic of the hybridizationprocess is shown in FIG. 4.

Another aspect of this invention is a method for separating by DMIPCtarget homoduplex and/or heteroduplex DNA fragments from a mixture ofhomoduplex and heteroduplex DNA fragments having the same base-pairlength, the heteroduplex fragments having at least one mismatch site.The method comprises the steps of applying the mixture of homoduplex andheteroduplex DNA fragments to a separation column containing separationmedia having a nonpolar, nonporous surface, the mixture of homoduplexand heteroduplex DNA fragments being applied in a first solutioncontaining a counterion and a DNA binding concentration of drivingsolvent. Then, while maintaining the media and the solution at atemperature which will locally denature the heteroduplex fragments atthe mismatch site thereof, separating desired homoduplex and/orheteroduplex DNA fragments from the separation media by contacting theseparation media with a second solution containing a counterion and aconcentration of driving solvent which has been predetermined toseparate the target homoduplex and/or heteroduplex DNA fragments fromthe separation media in separate fractions.

Thus, heteroduplexes and homoduplexes have been separated by HPLC usinggradient elution and “partially denaturing conditions” (U.S. Pat. No.5,795,976 to Oefner (1998)). However the separations achieved by Oefnerwere poorly resolved and not always reproducible.

Gradient elution MIPC under partially denaturing conditions achievesgood separations of homo and heteroduplexes, as described in U.S. patentapplication Ser. No. 09/129,105, filed Aug. 4, 1998. This reference andthe references contained therein are incorporated in their entiretiesherein. MIPC separates dsDNA fragments by base pair length undernon-denaturing conditions. However, under partially denaturingconditions, a structural component effects the separation as well.Therefore, homoduplexes and heteroduplexes separate from each other whenMIPC is conducted at partially denaturing temperature. The partiallydenaturing temperature varies with the sequence of any given DNAfragment. However, a preferred partially denaturing temperature isachieved between 50° C. to 70° C. a most preferred partially denaturingtemperature is 53° C. to 62° C., and an optimum partially denaturingtemperature is about 56° C. Liquid chromatographic separation ofhomoduplexes and heteroduplexes using an isocratic mobile phase has notbeen previously reported.

In this embodiment of the present invention, the hybridized dsDNAmixture is applied to a MIPC column comprising nonpolar separation mediaand the fragments are eluted with an isocratic mobile phase. Thedifference in retention time, shown in FIG. 31, of the heteroduplexesand homoduplexes is more than two minutes, a completely unambiguousseparation and a great improvement over a similar separation usinggradient elution. This wide separation can be used to advantage tocollect and isolate a heteroduplex as it elutes from the column. PCRamplification of the heteroduplex will provide sufficient pure materialfor sequence determination or other applications which require highpurity material.

In a preferred embodiment of the present invention, “kineticseparations” are used for mutation detection and utilize MIPC separationat a temperature which yields partial melting of mismatches. Thisembodiment achieves better resolution between heteroduplexes andhomoduplexes. When mixtures of DNA fragments are applied to an MIPCcolumn, they are separated by size, the smaller fragments eluting fromthe column first. However, when MIPC is performed at an elevatedtemperature which is sufficient to denature that portion of a DNAfragment which contains a non-complimentary base pair or polymorphism,then heteroduplexes separate from homoduplexes.

The “kinetic separation” is a method of separation performed underisocratic or near-isocratic conditions where the temperature conditionsare selected so that the hysteresis effect (illustrated in FIG. 32) isused to enhance the separation of heteroduplexes and homoduplex species.The temperature is selected so that the partially melted heteroduplexspecies does not interact with the stationary phase but travels throughthe column almost at the linear velocity of the mobile phase. Thehomoduplex interacts or is adsorbed by the column and elutes later.

During the cooling step of hybridization, the higher meltinghomoduplexes rapidly renature completely while the heteroduplexes,having been more extensively denatured during heating, renature slowly.Cooling while melting temperatures are fixed is equivalent

FIG. 32 illustrates the effect of higher temperatures on percentage ofdenaturation to maintaining a constant temperature and raising themelting temperature. Renaturation is kinetically slow beyond a criticaldegree of denaturation. That is, the higher the temperature used fordenaturation, the longer the time required for renaturation to occur.

One aspect of the present invention provides a method which can be usedto isolate mutations in a sample containing a relatively large amount ofwild type, wherein the concentration of the mutation can be below thelimits of detection by a detector. Alternatively, the invention providesa method for detecting mutations when the concentration of mutant DNA ina sample may be sufficient to detect, but the mutant DNA is not seenbecause it is obscured by the relatively large amount of wild type inthe sample. The invention takes advantage of the unique and surprisingattributes of MIPC and DMIPC to accomplish the objective of detectingmutations in such samples, wherein the wild type and mutant are known.

One aspect of this invention is a method for determining the presence orabsence of a homoduplex or a heteroduplex faction in a mixture ofhomoduplex and heteroduplex DNA fragments having the same base-pairlength, the heteroduplex fragments having at least one mismatch site.This procedure comprising the steps of applying the mixture ofhomoduplex and heteroduplex DNA fragments to a separation columncontaining separation media having a nonpolar, nonporous surface, themixture of homoduplex and heteroduplex DNA fragments being applied in afirst solution containing a counterion and a DNA binding concentrationof driving solvent. Then, while maintaining the media and the solutionat a temperature which will locally denature the heteroduplex fragmentsat the mismatch site thereof, separating desired homoduplex and/orheteroduplex DNA fragments from the separation media by contacting theseparation media with a second solution containing a counterion and aconcentration of driving solvent which has been predetermined toseparate the target homoduplex and/or heteroduplex DNA fragments fromthe separation media in separate fractions.

In the detection of mutations, the PCR primers are preferably selectedto yield fragments for which complete resolution of heteroduplexes fromhomoduplexes can be achieved by MIPC. Details for suitable primerselection are provided in copending U.S. patent application Ser. No.09/129,105 filed Aug. 4, 1998, the entire contents of which are herebyincorporated by reference.

In one preferred embodiment, the invention comprises a number of stepswhich eliminate any ambiguity regarding the presence or absence of aparticular mutant fragment in a sample when the sample contains a largeamount of wild type DNA relative to a putative mutation. These steps aredescribed hereinbelow.

The method for separating homoduplex and heteroduplex DNA fragments froma mixture of homoduplex and heteroduplex DNA fragments by DMIPCcomprises the steps of applying the mixture of homoduplex andheteroduplex DNA fragments to a separation column containing mediahaving a nonpolar, nonporous surface. The mixture of DNA fragments areapplied in a first solvent mixture containing a counterion and a DNAbinding concentration of driving solvent in a cosolvent. The desired DNAfragments are separated from the media by contacting the media with asecond solvent solution containing a counterion and a concentration ofdriving solvent in cosolvent which has been predetermined to selectivelyremove the desired fragments.

The desired fragments can be a homoduplex fraction. The concentration ofdriving solvent is selected to remove the homoduplex fraction, and thehomoduplex fraction is collected. The collected fractions can beamplified to obtain an increased ratio of heteroduplex relative tohomoduplex. Alternatively, the desired fragments can be a heteroduplexfraction, and the concentration of driving solvent is selected to removethe heteroduplex fraction, and the heteroduplex fraction is collected.

The method is particularly useful in the Sklar et al methods, that is,when the DNA sample contains a large background of wild type, the mutantDNA is below the limit of detection, the DNA sequences of the wild typeDNA and the mutant DNA are known, and the mutant DNA differs from wildtype DNA by at least one base pair.

In these separation methods, the retention time used in the separationof the homoduplex or heteroduplex fraction is determined from areference standard which can be obtained, for example, by separating astandard mixture of homoduplex and heteroduplex, having the same basepair sequence as the sample, by Matched Ion PolynucleotideChromatography. In the gradient and isocratic separation of DNAmixtures, and in the denaturing MIPC methods, the retention time used inthe separation of the homoduplex or heteroduplex fraction can bepreviously determined from a reference standard and is highlypredictable. The reference standard can be obtained in the denaturingMIPC methods, for example, by separating a standard mixture ofhomoduplex and heteroduplex, having the same base pair sequence as thesample, by Matched Ion Polynucleotide Chromatography and identifying theelution time or times for the desired fragments.

Since the base sequence of the sample wild type DNA and the putativemutation are known, standards of these materials are combined andhybridized. Hybridization is effected by heating the combined standardsto about 90° C., then slowly cooling the reaction to ambient temperatureover about 45 to 60 minutes. During hybridization, the duplex strands inthe sample denature, i.e., separate to form single strands. Uponcooling, the strands recombine. If a mutant strand was present in thesample having at least one base pair difference in sequence than wildtype, the single strands will recombine to form a mixture ofhomoduplexes and heteroduplexes. In this manner, a standard mixture ofhomoduplexes and heteroduplexes is formed as depicted schematically inFIG. 4. The standard mixture contains the same homoduplexes andheteroduplexes present in a sample which contains a putative mutation,albeit not in the same ratio. This standard mixture cannot be separatedby MIPC under normal conditions, since the heteroduplex and homoduplexhave the same base pair length. However, when MIPC is performed at atemperature sufficiently elevated to selectively and partially denaturea heteroduplex at the site of base pair mismatch (DMIPC), the partiallydenatured heteroduplex will separate from a homoduplex having the samebase pair length. Therefore, the hybridized standard mixture is appliedto a MIPC column and a separation is performed under DMIPC conditions.The chromatogram so produced shows a separation of the homoduplexes andheteroduplexes as shown in FIG. 5. The retention times of the separatedhomoduplex and heteroduplex standards can then be used to predict theretention times of putative mutations having a concentration too low tobe detected by a detector. Alternatively, the retention times of theseparated homoduplex and heteroduplex standards can then be used topredict the retention times of putative mutations in samples wherein themutation signal is obscured by the wild type signal.

Having determined the retention times of the standards, a samplecontaining a putative mutation is amplified using PCR to increase thetotal quantity of sample. Since the sequence is known, primers can bedesigned to maximize the fidelity of replication and minimize theformation of reaction artifacts and byproducts. Approaches to primerdesign and PCR optimization for mutation detection by DMIPC arediscussed in co-pending U.S. patent application Ser. No. 09/129,105filed Aug. 4, 1998. However, wild type and mutant DNA strands in asample have a nearly identical base sequence. A mutation may containonly one base pair difference compared to wild type. Therefore, primerscannot be designed to selectively anneal to, and preferentially amplifythe mutant strand in the presence of wild type. Therefore, when such asample is amplified using PCR, the ratio of mutant to wild type in theamplified product will be the same as in the original sample.

When the amplified sample is analyzed using MIPC a single major peakwill be seen in the resulting chromatogram. This peak represents thecombined wild type and mutant DNA, if the latter is present. Noseparation is achieved because the mutant and wild type DNA have thesame base pair length. Therefore, the amplified sample is hybridized andanalyzed under partially denaturing conditions by DMIPC. However, theheteroduplex corresponding to the putative mutation, if present, willnot be seen by the detector either because its concentration is belowthe detection limits of the detector or because the ratio of wild typeto putative mutation is very large so that the wild type homoduplex peakobscures the heteroduplex peak.

In either case, the heteroduplex corresponding to the mutant DNA in theoriginal sample need not be seen as a chromatographic peak to bedetermined. Having previously identified the retention time of theheteroduplex standard, the mobile phase is “blind collected” from thecolumn at the expected retention time.

The “blind collected” mobile phase described hereinabove preferably isconcentrated, e.g., by evaporation of the mobile phase. If a mutationwas present in the original sample, the residue will now be enriched inthe heteroduplex. This heteroduplex enriched residue is amplified againby PCR and the products are hybridized. The hybridized products of thesecond PCR amplification will now contain an increased amount ofheteroduplex relative to homoduplex. A chromatogram showing the resultsof this process is depicted in FIG. 6. The evaporation can be effectedwith standard and conventional DNA solution evaporation equipment, forexample, the SPEEDVAC evaporator (Model UCS 100 Universal Speed Vacsystem, Savant Instruments, Inc, Hayward, Calif.).

The collected fragments can be amplified by conventional PCR or cloningand further analyzed by sequencing and restriction digestion. The cycleof separation and amplification can be repeated, if needed, to increasethe amount of a desired fraction available for subsequent analysis.

The steps comprising the method of the invention were designed to enrichthe sample in heteroduplex in order to enable the detection of mutationswhich would normally go undetected. The steps of the method of theinvention can be reiterated a plurality of times to increase the purityand quantity of heteroduplex to any desired level. The increased amountof heteroduplex compared to homoduplex obtained in this manner can bedescribed by an “enhancement factor”. The “enhancement factor” isdefined herein as the increase in the ratio of heteroduplex tohomoduplex compared to the ratio of heteroduplex to homoduplex in theoriginal hybridized sample, wherein the increase results from theimplementation of the method of the invention. The “enhancement factor”depends on the number of iterations performed and can range from 10 tomore than 1,000.

After the final iteration, the PCR product is hybridized and analyzed byDMIPC. If the original sample contained a mutation, the concentration ofheteroduplex or its concentration relative to wild type, will now besufficient to detect. The DMIPC chromatogram will, therefore, show apeak having the retention time of the standard heteroduplex. In thisevent it can be concluded unambiguously that a mutation was present inthe original sample.

As a further confirmation of the identity of the mutation, an aliquot ofstandard heteroduplex can be mixed with an aliquot of the heteroduplexenriched sample. A DMIPC chromatogram of this mixture will show anincrease in the area of the heteroduplex peak, compared to the area ofthe heteroduplex enriched sample peak alone.

Additionally, the purification and enrichment method described abovewill provide sufficient heteroduplex for determination of its base pairsequence. Sequencing will provide further confirmation of the identityof the mutation.

If, after performing a plurality of iterations according to the methodof the invention as described above no heteroduplex peak is seen in theDMIPC chromatogram, then it can be safely concluded that the originalsample did not contain a mutation.

Denaturing gradient gel electrophoresis techniques which can separatehomoduplexes from heteroduplexes cannot be used as an alternative toDMIPC. Although samples can be recovered from gels with difficulty,blind collection is not possible because the mobility of a DNA fragmentin a gel is not constant. Therefore, its position cannot be reliablypredicted. In addition, the shape of DNA fragment bands in gels areoften irregular, further complicating sample recovery and makingdetection uncertain. An additional problem is the fact that gels takemany hours to develop, making it impractical for routine use.

On the other hand, the highly predictable nature of the retention timesdetermined from DMIPC separations makes this method uniquely suited tomutation detection if blind collection is required. The use of DMIPC forthe purpose of mutation detection as described in this application hasnot been previously reported.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

FIG. 7 is a cross-sectional view of a spin vial separation device ofthis invention. This system uses a standard laboratory centrifuge torapidly pass liquids through the separation media. The system uses astandard cylindrical centrifuge vial or eluant container 142 into whicha separator tube or cylinder 144 is inserted. The separator cylinder canhave a cylindrical body 146, open at top end 148 and bottom end 150, andsized to fit within the vial 142. The upper end 148 has an outwardlyextending upper flange 152 which is sized to rest on the upper rim 154of the cylindrical vial 142. The lower end 150 has an inwardly extendinglower flange 156 which is sized to support the separation unit 158.

The separation unit comprises a porous support disk 160 which rests onflange 156, an optional outer cylinder 162 within which the separationmedia 164 is positioned. The separation unit can also comprise anoptional upper porous disk 166 to prevent disruption of the separationmedia and an optional ring 168. The optional ring 168 preferably has aslightly elastic or yielding composition and an outer diameter which issized to establish a frictional engagement with the inner wall ofcylinder 146. The ring 168, when pressed against the disk 166, holds thedisk in place during use of the column.

The separation media is a unique aspect of this invention. The surfacesof the media must be nonpolar surface and must be free of any traces ofmetal contaminants such as multivalent metal ions. The media can be inthe form of beads; monoliths; a bundle of capillary tubing or an objectwith parallel capillary passageways having one set of ends open to theupper separation chamber 170 and the other set of ends open to the lowerseparation chamber 172. The media surfaces can be porous or nonporous.However, to effect rapid and precise separations, nonporous mediasurfaces are preferred. Examples of porous media are described incopending, commonly assigned U.S. patent application Ser. No. 09/081,039filed May 18, 1998. Examples of the preferred nonporous media aredescribed in copending, commonly assigned U.S. patent applications Ser.No. 09/183,123; filed Oct. 30, 1998 and Ser. No. 09/183,450 filed Oct.30, 1998.

These separation media can be beads or other structures described above.The preferred separation media are beads which are made of nonpolarmaterials such as the organic polymers described in U.S. patentapplications Ser. No. 09/183,123 or inorganic polymer beads which havebeen treated to end-block or coat their polar groups. The optimum beadsare organic polymer beads such as styrene-divinyl benzene polymer beadswhich can have optional alkyl substitutions described in U.S. patentapplications Ser. No. 09/183,123.

The beads are preferably made from polymers, including mono- and divinylsubstituted aromatic compounds such as styrene, substituted styrenes,alpha-substituted styrenes and divinylbenzene; acrylates andmethacrylates; polyolefins such as polypropylene and polyethylene;polyesters; polyurethanes; polyamides; polycarbonates; and substitutedpolymers including fluorosubstituted ethylenes commonly known under thetrademark TEFLON. The base polymer can also be mixtures of polymers,non-limiting examples of which include poly(styrene-divinylbenzene) andpoly(ethylvinylbenzene-divinylbenzene). The polymer can beunsubstituted, or substituted with a hydrocarbon such as an alkyl grouphaving from 1 to 1,000,000 carbons. In a preferred embodiment, thehydrocarbon is an alkyl group having from 1 to 24 carbons. In morepreferred embodiment, the alkyl group has 1-8 carbons.

The separation media surfaces must-be free from multivalent metalcations in order to obtain the most effective separations.

The separations of materials using the device of FIG. 7 are demonstratedin the Examples presented hereinbelow. In general, they are achieved bythe following sequence of steps.

1. The mixture of a double-stranded oligonucleotides, double-strandedDNA fragments, single-stranded DNA fragments, RNA, plasmids or the liketo be separated is diluted in an aqueous solution containing acounterion and a low concentration of driving solvent.

2. The diluted mixture is placed in the chamber 170 and the separationdevice with the liquid is placed in a standard laboratory centrifuge andspun until all of the free liquid has passed into the chamber 172. Theinner cylinder 144 is removed from the vial, and the contents of chamber172 are discarded. The materials to be separated bind to the separationmedia in this step.

3. A second aqueous solution containing counterion and a second higherconcentration of driving solvent is prepared and placed in the chamber170. The driving solvent concentration is calculated to be the amountwhich will remove all undesired smaller size materials from theseparation media. The precise concentration of driving solvent isdetermined from -a standard table or curve, or it can be calculated,using values determined in a calibration of the media made using adouble-stranded DNA (dsDNA) ladder or standardized enzyme digest.

4. The separation device is spun in a centrifuge until all of the freesecond solution has passed into the chamber 172. The inner cylinder 144is removed from the vial, and the contents of chamber 172 are removed.This step removes from the separation media contaminants and smallersize fragments.

5. A third aqueous solution containing counterion and a third higherconcentration of driving solvent is prepared and placed in the chamber170. The third higher concentration of driving solvent concentration iscalculated to be the amount which will remove a desired larger-sizedmaterial from the separation media. The precise concentration of drivingsolvent is determined from a standard table or curve, or it can becalculated, using values determined in a calibration of the media madeusing a double-stranded DNA (dsDNA) ladder or standardized enzymedigest.

6. The separation device is spun in a centrifuge until all of the freethird solution has passed into the chamber 172. The inner cylinder 144is removed from the vial, and the contents of chamber 172, containingthe desired larger-sized fraction or fractions, are removed for furtherprocessing. This step removes from the separation media the desiredmaterials.

Obviously, the vial 142 can be replaced between steps or cleaned betweensteps to prevent contamination of the product fraction or fractions.

The concentration of driving solvent in the third solution can beselected to remove a single fragment size or a range of fragment sizes.

Steps (5) and (6) can be repeated with successive greater concentrationsof solvent to remove a series of successively increasing fragment sizes.

It will be readily apparent to a person skilled in the art that othervariations can be applied to remove a series of purified fractions inmuch the same manner as is shown above and illustrated in the Examplesand Figures of this application.

Examples of suitable organic driving solvents include alcohol, nitrile,dimethylformamide, tetrahydrofuran, ester, ether, and mixtures of one ormore thereof, e.g., methanol, ethanol, 2-propanol, 1-propanol,tetrahydrofuran, ethyl acetate, acetonitrile. The most preferred organicsolvent is acetonitrile. The counterion agent is preferably selectedfrom the group consisting of lower alkyl primary amine, lower alkylsecondary amine, lower alkyl tertiary amine, lower trialkyammonium salt,quaternary ammonium salt, and mixtures of one or more thereof.Non-limiting examples of counterion agents include octylammoniumacetate, octadimethylammonium acetate, decylammonium acetate,octadecylammonium acetate, pyridiniumammonium acetate,cyclohexylammonium acetate, diethylammonium acetate, propylethylammoniumacetate, propyldiethylammonium acetate, butylethylammonium acetate,methylhexylammonium acetate, tetramethylammonium acetate,tetrapropylammonium acetate, tetrabutylammonium acetate,dimethydiethylammonium acetate, triethylammonium acetate,tripropylammonium acetate, tributylammonium acetate, tetraethylammoniumacetate, tetrapropylammonium acetate, tetrabutylammonium acetate, andmixtures of any one or more of the above.

Examples of suitable counterion agents include an anion, e.g., acetate,carbonate, bicarbonate, phosphate, sulfate, nitrate, propionate,formate, chloride, perchlorate, or bromide. The most preferredcounterion agent is triethylammonium acetate or triethylammoniumhexafluoroisopropyl alcohol.

FIG. 8 is a cross-sectional view of a vacuum tray separation device ofthis invention, and FIG. 9 is a top view of the separation tray of FIG.8. The separator tray 200 is a single plate with rows and columns oftubular separation channels 202, preferably having regular, repeatedspacings between the rows and columns for indexing the spacings. Thedimensions of the tray 200 and separation channels can correspond andmatch the dimensions of standard multiwell plates such as the 96 cavitymicrotiter plate.

The multi-channel plate 200 is supported on support flange and vacuumseals 204 formed in the internal cavity of an upper plate 206 of thevacuum assembly 207. The vacuum assembly 207 further comprises a vacuumcavity 208 defined by housing 210. The upper plate 206 positioned on thehousing 210 by locating pins 212, and the upper plate 206 and thehousing 210 have a sealed engagement with the seals 204. The housing 210has an exhaust outlet channel 214 communicating with the vacuum chamber208 and with a vacuum conduit 216 and vacuum valve 218. The vacuumconduit 216 and vacuum valve 218 communicate with a vacuum source (notshown).

A multiwell collection plate 220 is supported in the vacuum chamber 208.The multiwell collection plate 220 is a single plate with rows andcolumns of separation channels 222, preferably having regular, repeatedspacings between the rows and columns for indexing the spacings. Thedimensions of the tray 220 and collection channels can correspond andmatch the dimensions of standard multiwell plates such as the 96 cavitymicrotiter plate. The collection plate 220 is held in a position whichaligns each of the collection wells 222 with a corresponding separatingchannel 202 of the separation plate 200 so each well 222 can collectliquid falling from the corresponding separation channel 202.

FIG. 10 is a cross-sectional view of the separation tray of FIG. 9 takenalong the line A—A. The separation, channels 202 each have an evenlyspaced upper cavity 224, separation media 226 and a liquid outlet 228.

FIG. 11 is an enlarged view of the separation components of theseparation tray of FIG. 10. The bottom of the separation cavity 224supports a porous disk 230, which in turn supports separation media 232.An optional containment disk 234 rests on the separation media 232, andthe containment disk 234 can be optionally held in place by frictionring 236 or an equivalent device.

The separation media 232 can be the same nonpolar media as describedabove with respect to media 122 in FIG. 3.

The separation vial components 142 and 146 of FIG. 7 and the plates 200and 220 in FIGS. 8-11 are made of a material which does not interferewith the separation process such as polystyrene, polypropylene, orpolycarbonate. The upper plate 206 and housing 210 can be made of anymaterials having the requisite strength such as a rigid organic polymer,aluminum, stainless steel or the like. The vacuum chamber walls arepreferably coated with Teflon film. The vacuum conduit and valve canalso be made of Teflon coated aluminum or the like.

The separations of materials using the device of FIGS. 8-11 are achievedby the following sequence of steps.

1) A mixture of double-stranded oligonucleotides, single-stranded DNAfragments, single-stranded DNA fragments, RNA, plasmids or the likecontaining a component to be separated from the mixture to be separatedis diluted in an aqueous solution containing a counterion and a lowconcentration of driving solvent.

2) The diluted mixture is placed in one of the chambers 202 of the fullyassembled vacuum device. The other chambers 202 are filled with othermixtures to be separated by the same procedure.

3) Vacuum is applied to the vacuum chamber 208 by opening vacuum valve218 until all of the liquid from the mixtures contained in each chamberhas collected in chambers 222. The vacuum device is disassembled, andthe contents of chambers 222 are discarded. The materials to beseparated bind to the separation media 232 in each chamber 202 in thisstep.

4) The vacuum apparatus and plates are reassembled, and a second aqueoussolution containing counterion and a second higher concentration ofdriving solvent is prepared and placed in the chambers 202. The drivingsolvent concentration is calculated to be the amount which will removeall lower molecular weight material from the separation media. Theprecise concentration of driving solvent is determined from a standardtable or curve, or it can be calculated, using values determined in acalibration of the media made using a double-stranded DNA (dsDNA) ladderor standardized enzyme digest.

5) Vacuum is applied to the vacuum chamber 208 by opening vacuum valve218 until all of the liquid from the mixtures contained in each chamberhas collected in chambers 222. The vacuum device is disassembled, andthe contents of chambers 222 are removed. The lower molecular weight andsmaller size materials are removed from the column and collected, andthe larger base-pair length materials remain on the separation media 232in this step.

6) The vacuum apparatus and plates are reassembled, and a third aqueoussolution containing counterion and a second higher concentration ofdriving solvent is prepared and placed in the chambers 202. The drivingsolvent concentration is calculated to be the amount which will remove adesired larger-sized material or materials from the separation media.The precise concentration of driving solvent is determined from astandard table or curve, or it can be calculated, using valuesdetermined in a calibration of the media made using a double-strandedDNA (dsDNA) ladder or standardized enzyme digest.

7) Vacuum is applied to the vacuum chamber 208 by opening vacuum valve218 until the liquid from the mixtures contained in each chamber hascollected in chambers 222. The vacuum device is disassembled, and thecontents of chambers 222 are removed. The larger-sized materials areremoved from the chambers and collected.

The concentration of driving solvent in the third solution can beselected to remove a single fragment size or a range of fragment sizes.This procedure can be elaborated by choosing a driving solventconcentration in the third aqueous solution which removes all materialsbelow a target size and following it with a repeated sequence with adriving solvent concentration which will remove the specific sizematerials of the target size.

Obviously, the plate 220 can be replaced between steps or cleanedbetween steps to prevent contamination of the product traction orfractions.

These steps can be repeated with successive greater concentrations ofsolvent to remove a series of successively increasing fragment sizes.

It will be readily apparent to a person skilled in the art that othervariations can be applied to remove a series of purified fractions inmuch the same manner as is shown above and illustrated in the Examplesand Figures of this application.

MIPC is uniquely suited to the process of nucleic acid purification,whether the fragments are tagged or untagged by exogenous moieties (i.e.fluorescent dyes, biotin, etc.). By applying MIPC conditions thatisolate fragments on the basis of size, sequence, or both, the highestpossible purity is obtained for nucleic acids. This level of performanceis achieved through tight control over chemical/eluent conditions, aswell as using appropriate matrix materials. With separations made withthe devices and methods of this invention, DNA fragments can be producedwhich are more suitable for cloning than can be obtained by gelelectrophoresis, for example, Hecker, Karl H. et al, “Optimization ofcloning efficacy by pre-cloning DNA fragment analysis”, Biotechniques,26(6):216-222 (February, 1999).

Since MIPC is a chemical process, nucleic acid purifications can beachieved at any pressure. This is especially valuable for size-basedpurification of PCR products and restriction digests at ambient pressureas shown in the Examples 3 and 4, and FIGS. 22-27 presented hereinafter.It is equally valuable for size-based (Marino, et al, Electrophoresis,1998, 19, 108-118) and sequence based purification of PCR products athigh pressure prior to sequencing as shown in Example 1, and FIGS. 12-21hereinbelow. It can also be applied for size-based purification ofplasmid restriction digests prior to cloning. See US ProvisionalApplication Serial No. 60/130,700 filed Apr. 23, 1999, incorporatedherein in its entirety.

The general process of obtaining purified nucleic acids are independentof pressure conditions. Whether operating at high or low pressures,purification is characterized by the following processes:

1. Trapping the nucleic acid(s) of interest on the DNA Chromatographymatrix under appropriate DNA Chromatography conditions.

2. Exposing the trapped nucleic acid(s) to chemical and/or thermalconditions which quantitatively release those components selected forremoval.

3. Collecting the removed components of interest for later use(optional).

4. Detecting the removed components (optional).

5. Exposing the remaining trapped nucleic acid(s) to chemical and/orthermal conditions which quantitatively release the remaining nucleicacid(s).

6. Collecting the released nucleic acid(s) of interest (optional).

7. Detecting the released nucleic acid(s) of interest (optional).

The following are examples of the application of DNA Chromatography fornucleic acid purification:

It is known that current technologies used for PCR purification havesignificant problems. One of the single biggest problems with thisapplication for current technology is the incomplete removal of primers,primer dimers or other non-specific amplification products. Throughcompetition, these “background” components confound many downstreammolecular biology tasks (such as sequencing and cloning, just to name acouple). As one example, these problems are particularly exasperating insequencing when using the industry-standard approach of dye-labeledterminators in cycle sequencing. When using dye-labeled terminators, thepurification of PCR-generated templates must provide exceptionally hightemplate recoveries and template purity. This means there must becomplete removal of potentially interfering contaminants (amplificationprimers and primer dimers, non-specific amplification products, dNTPs,Taq) which doesn't always occur with current silica-based technology.

Other downstream problems also occur when cloning is being performed, asit can lead to the cloning of undesired fragments. These cloningproblems are outlined in Hecker, Karl H. et al, supra, the entirecontents of which are incorporated by reference.

The purification approach taken by major producers of PCR cleanuptechnology is based upon the adsorption of nucleic acids (ssDNA, dsDNA,RNA) to silica gel. Once the silica gel is treated with a high-saltsolution, the nucleic acids adsorb to the silica gel via an exchangemechanism. The nucleic acids are then desorbed by applying a low-salteluent. This works very well for clean DNA, but has problems with othercomponents. The chemical processes occurring on the silica gel surfaceare not tightly controlled, so one is dependent upon radical changesoccurring evenly across the particle surface. The lack of efficientprimer, primer dimer and non-specific amplification product removal iswell known. Example 5 illustrates removal of PCR byproducts.Furthermore, the (typically) porous surface of the silica gel can trapthose contaminants that most require removal.

The materials used currently in the MIPC column matrix of thisinvention, as well as other materials suitable for MIPC (as one example,larger polymeric particle sizes of nonporous reverse-phase materials),are known to have an exceptionally high capacity and selectivity forlong-chain nucleic acids. By applying a suitable pairing ion, and thenchanging nothing other than the acetonitrile concentration (or any othersuitable solvent, such as an alcohol), the quantitativeadsorption/desorption of varying lengths of short-and long-chain nucleicacids are essentially turned on and off. Furthermore, since the matrixis made of a nonporous polymeric material, there is no opportunity forinterlopers (dNTPs, primers, primer dimers, non-specific amplificationproducts) to get trapped and become problematic downstream. In essence,the matrix materials we possess (nonporous polystyrene-divinylbenzene,either unalkylated or alkylated), are perfectly suited to thepurification of PCR products prior to the most demanding molecularbiology applications. Also suitable are nonporous polymeric or modifiedsilica materials which has been manufactured or purified in a mannerwhich produces surfaces which are free of contamination. These can be inthe form of beads, monoliths, channels, capillary or planar surfaces.The polymeric surfaces can be provided by non-alkylated and alkylatedmaterials including polystyrene, divinylbenzene,hydroxyethylmethacrylate, and other nonionic polymers. Polymers having anegative charge may also be used provided the charged groups areprotonated to produce a neutral surface, i.e., carboxylic acid. Anexample of this is illustrated by Example 1. When trying to dosize-based cloning from a pool of fragments (cDNA fragment pools,fragment pools from restriction digestion of plasmids/artificialchromosomes/genomic DNA, etc), the efficiency of fragment ligation intothe vector (and hence cloning efficiency) is biased towards smallerfragments. As an extension of the identical principles applied forpurification of PCR products, one can perform fractionations of fragmentpools on the kits. By applying gradually increasing concentrations ofacetonitrile and analyzing the collected fractions with a suitable260/280 detector, an extremely rapid process is created that effectivelyreplaces smearing the fragment pools out on a gel and slicing the sizerange of interest out with a razor blade. This general process has beenshown for high-pressure sizing of fragments prior to cloning, and willtranslate directly into low-pressure formats.

It is known that many silica-based and anion exchange-based purificationkits for clones (this is particularly true for mini-preps of ≦20 μgplasmid) have difficulty with purifications from TB (terrific broth),due to the higher copy number of plasmids present (relative to LBbroth). We know that DNA Chromatograpy matrices have higher capacitiesthan typical silica-gel-based preps (i.e. QiaGen miniprep). Furthermore,it is highly likely that since our separation mechanism is akin to anionexchange, DNA Chromatography-derived purities will match or exceed thepurities of competitor's premier (and significantly more expensive)“ultra-high-purity” anion exchange product, thus representingsignificant cost savings at the same time.

The following is a protocol for this procedure.

First, Plasmid DNA is released from E. Coli (or, ssDNA from M13 phage).Alkaline lysis of E. Coli is a classic first step for the isolation ofplasmids from E. Coli. To achieve this, the cells are centrifuged toseparate them from the supernatant/growth medium (either Luria-Bertani(aka LB) or terrific broth (aka TB) medium). Once pelleted, the cellsare resuspended (often with glucose/EDTA/Tris, aka GET) and then lysedwith NaOH/SDS. This must be done gently, so as to not introduce smallerpieces of sheared chromosomal DNA. The resultant solution should befairly clear and viscous. This solution is neutralized with anappropriate buffer (often potassium acetate), thus generating a whiteprecipitate which contains cellular debris, SDS, chromosomal DNA, andsome proteins. The plasmid DNA (along with various soluble lipids,various proteins, carbohydrates, salts, RNA) remains in solution. Thelysate solution is typically clarified via centrifugation. Thisclarified lysate, which contains the plasmid or ssDNA, is thenintroduced to the DNA Chromatography matrix.

Once introduced to the matrix, a wash procedure will take place. Thiswould likely be a TEAA solution with a higher concentration ofacetonitrile (i.e. 15% acetonitrile). This should remove even the mosthydrophobic contaminants, and leave the plasmid intact on the resin.Once washed with this solution, the plasmid would be eluted with aminimum amount of eluting buffer (25% acetonitrile, higher for largerplasmids). In the case of ssDNA from M13 phage, it may be necessary togo to a different alkyl ammonium ion-pairing reagent so that size-basedseparation is maintained. The collected plasmids/M13 phage (as anoption, the solvents can be removed via evaporation) are then availablefor any downstream application.

These are extremely large DNA constructs (typically >100 kb) which oftenmust be purified for a number of reasons (sequencing, sub-cloning, etc).Note that there are a vast number of procedures available for therelease of these constructs from their respective mediums (i.e.bacteria, yeast, other cells). In any event, once the constructs arepresent in a clarified lysate medium (much like plasmids and M13 phage,above), they must have a suitable quantity of ion-pairing reagent addedto them so they can trap onto the DNA Chromatography matrix. Oncetrapped, a wash solution with an appropriate concentration of organicsolvent (acetonitrile, alcohol, etc) is applied to remove contaminatingspecies, leaving the larger DNA constructs on the matrix. Since theseconstructs are quite large, they will likely require a fairly highconcentration of acetonitrile (or other suitable solvent) to releasethem from the matrix. Once they are collected (as an option, thesolvents can be removed via evaporation) the constructs are availablefor any downstream application.

MIPC is well suited to purification of oligonucleotides and longerprobes, both of which are ssDNA constructs. Oligonucleotides and probes(either labeled or unlabeled) require quality control and/orpurification prior to use—either directly after in-house synthesis, butalso in cases where they are ordered from external suppliers (and areassumed to have gone through these special purification measures). It ispossible to perform these purifications in a high-pressure format, as isdemonstrated in detail in Example 1 and Example 6.

It is also possible to perform these analyses in a low-pressure formatas well. As in the case of the nucleic acids mentioned above, the ssDNAconstructs are combined with a suitable concentration of an appropriateion-pairing reagent that allows size-based discrimination of the DNA.

A standard means of performing genotyping involves the use of primerextension. An oligonucleotide is annealed which flanks the 5′ region ofinterest on a template. Nucleotides are added in standard proportions,except for one of them being a terminating dideoxynucleotide (ddNTP). Anappropriate high-fidelity polymerase, such as Pfu, is added and thenucleotides are added on to the template, effectively extending theprimer to the point where the ddNTP is incorporated, thus halting theprimer extension process. The length of the resultant extension productthus indicates the identity of the base where extension terminated. Thisextension may only occur for one base, or it may go on until the basecomplementary to the terminator is encountered. In any event, it ispossible to then denature the extended primer from the template, andthen size the extended primer to determine an accurate genotype for thetemplate.

In the context of DNA Chromatography, it is possible to use temperature(>75 C.) to completely denature the extended primer from its template.Upon introduction to the pre-heated matrix, the (ion-paired) templateand (ion-paired) extended primer will remain adsorbed on the surfaceuntil a suitable concentration of organic solvent (acetonitrile,alcohol, etc) is introduced that selectively desorbs only the(significantly shorter) primers and primer extension products (possiblyfor collection), thus leaving the original template behind on thematrix. This is an example where smaller products are collected first,and larger (unnecessary) products are left behind.

Once the primer extension products are selectively released, they can besized directly on the DNA Chromatography matrix ( Hoogendoom, B., et al“Genotyping single nucleotide polymorphisms by primer extension and highperformance liquid chromatography”, Human Genetics, 1999, Vol. 104, No.1, pp. 89-93. If the purified primer extension products are collected,they can also be sized by other means such as gel electrophoresis ormass spectrometry.

When the purification procedures noted above are applied, it is possibleto detect the purified products either by UV absorbance, or byfluorescent intensity. In the case of high- or moderate-pressurepurifications occurring in a conventional liquid chromatography format,this detection occurs on-line with a suitable detector (this has beendescribed in detail in other documents). When these processes occur in alow-pressure format, it is also possible to detect the purified productsin an on-line format. However, a straightforward means of detecting thepurified products is in an off-line format (either in a cuvette-basedsystem, or by a suitably designed well-plate reader).

Independent of the detection means described above, when the nucleicacids are discretely detection after purification, a replacement forgel-based purifications has been effected. As an illustration of thisconcept, an example is given below for the detection of (low-pressure)purified PCR products prior to dye-terminator sequencing.

When a PCR product has been purified and collected into an appropriatewell-plate, it will require spectral analysis. The reasons for this aretwo-fold: successful DNA sequencing (particularly dye terminator cyclesequencing) requires a fair amount of control over the amount of DNAintroduced for sequencing; the amount of protein impurities must bemeasured, to ensure there are no competing enzymatic reactions. Toachieve these measurements, the purified DNA must be measured viaabsorbance at 260 nm (for DNA measurement) and at 280 nm (for proteinmeasurement). This can be done by collecting the eluted DNA in aUV-transparent well-plate. Note that we must ensure the absence ofphotometric “crosstalk” between the wells, and that path lengths are“adjustable” to reflect different eluant volumes. The spectrophotometricrequirements at each wavelength are very important. When operating atmaximum DNA capacity, 10 ug DNA/50 uL=200 ng/uL: when operating undertypical conditions: ˜1 ug/50 uL=40 ng/uL. One can assume A nominal pathlength of 0.2 cm (50 μL in polyfiltronics' UVMAX plate). Absorbance of1.0 at 260 nm (A260=1)=50 ng/uL DNA, with 1.0 cm path length∴A260=0.2for 50 ng/uL DNA in a 0.20 cm path length. If the conditions noted aboveare the case, then the following absorbance scale (minus background)must be achievable with the A260 measurements at a 0.20 cm path length:

Capacity (10 μg DNA/50 μL = 200 ng/μL): A260 = 0.8 Typical (1 μg DNA/50μL = 20 ng/μL): A260 = 0.08 Low End (50 ng DNA/50 μL = 1 ng/μL): A260 =0.004

For the purposes of protein “quantitation” and measuring DNA purity, itwill be necessary to take measurements at 280 nm. Ratios of theA260/A280 measurements will be used for this task. Using the absorbancescale above as a guide, and knowledge that A260/A280 ratios must beabove 1.8 for typical sequencing reactions, the following must beachievable at 280 nm.

Capacity (200 ng/μL): A280 = 0.440 Typical (20 ng/μL): A280 = 0.044 LowEnd (1 ng/μL) A280 = 0.0022

There are many procedures within molecular biology that require thecomplementary (or, near complimentary) hybridization of a probe (orprobes) to a test strand (or strands) of DNA or RNA. Once the probe (orprobes) have been hybridized, the hybridization event is then detectedby a number of means. To detect the hybridization event, it is possibleto examine the signal from a reporting moiety attached to the probe(radioactive probes, fluorescent probes, etc), to examine a mobilityshift in a gel electrophoresis experiment, or the like.

It is also possible to detect hybridization events directly by use ofDNA Chromatography. Once a probing event has occurred, and any othersteps are taken afterwards (i.e. extension of an annealed primer,displacement of the probe for analysis, etc), the resultantsingle-stranded structure (which will be smaller than the template it isinterrogating) can be denatured directly from the template and detectedeither on-line (i.e. UV absorbance, fluorescence, mass spectrometry) orin an off-line manner (i.e. MALDI-TOF-MS). Demonstrative examples aregiven below for these types of operations and analyses. Note that all ofthe processes described herein are chemical processes, and can beperformed in a high-pressure or low-pressure environment. Furthermore,all of these processes can be performed at any scale (including micro-or nano-scale).

A standard means of performing genotyping involves the use of primerextension. An oligonucleotide is annealed which flanks the 5′ region ofinterest on a template (either DNA or RNA). Nucleotides are added instandard proportions, except for one of them being a terminatingdideoxynucleotide (ddNTP). An appropriate high-fidelity polymerase isadded (i.e. Pfu), and the nucleotides are added on to the template oneby one, effectively extending the primer to the point where the ddNTP isincorporated—thus halting the primer extension process. The length ofthe resultant extension product thus indicates the identity of the basewhere extension terminated. This extension may only occur for one base,or it may go on until the base complementary to the terminator isencountered. In any event, it is possible to then denature the extendedprimer from the template, and then size the extended primer to determinean accurate genotype for the template.

In the context of DNA Chromatography, it is possible to use temperature(i.e. >75° C.) or other means to completely denature the extended primerfrom its corresponding template (Hecker, Karl H. et al, supra, ). Forexample, upon introduction to the pre-heated matrix, the (ion-paired)template and (ion-paired) extended primer will remain adsorbed on thesurface until a suitable concentration of organic solvent (acetonitrile,alcohol, etc) is introduced that selectively desorbs only the(significantly shorter) primers and primer extension products, thusleaving the original template behind on the matrix. This is an examplewhere smaller products are purified and collected first, and larger(unnecessary) products are left behind.

It is also possible to analyze these extended products directly on-lineby DNA Chromatography. By applying proper chemical conditions foraccurate sizing of ssDNA, the extension products are eluted in such amanner as to give information about where the extension process wasterminated, and hence indicate the identity of the terminatingnucleotide. For example, an 18-mer primer is annealed (underlined) 5′ toa variant site (let us say that the variant, in bold, is either a C or aT), and the terminating nucleotide added is ddGTP. The possible variantsare shown as examples below.

Variant 1: GTCATCGAATCCATGCTACAC

Variant 2:GTCATCGAATCCATGCTATAC

Once primer extension occurs, the products are either 19 bases long (inthe case of a C variant) or are 21 bases long (in the case of a Tvariant, and extension ceases when the next C nucleotide isencountered).

If the subject is homozygous for Variant 1, then only one peak (for 19mers) will be detected. If they are homozygous for Variant 2, then onlyone peak (for 21 mers) will be detected. If they are heterozygous, thentwo peaks (19-mers and 21-mers) will be detected. In effect, by applyingdenaturing conditions and proper ssDNA sizing operations, all of thesedeterminations can be performed in a manner that is automated.Furthermore, since DNA Chromatography is a quantitative process, thistranslates directly to genotyping, such that “allele counting” can occurin a straightforward manner when studying population genetics.

A further presentation of these is presented in TABLE A wherein the (C)represents the single nucleotide polymorphism (SNP) to be detected, (Crather than A)

TABLE A 1.

2.

3.

4.

It is also possible to analyze these extension products directly by asuitable “real time”. mass spectrometric detection scheme, which willdetect these species as they emerge from the DNA Chromatography system.This would be applicable when all of the potential terminatingnucleotides are added at one time (ddATP, ddCTP, ddGTP, ddTTP), thusgiving mass differences between the eluted extension products. However,this would allow the direct identification of the variant nucleotide ina single step. Using the example above, if the subject were heterozygous(Variant 1 and Variant 2 present), two 19-mer primer extension productswould be generated. However, their masses would differ by(5517_(primer mass)+329_(ddGTP for C allele))_(Variant 1)−(5517_(primer mass)+313_(ddATP for T allele))_(Variant 2)=16mass units.

Regardless of whether the mass spectrometer is “off-line” (MALDI−TOF) or“on-line” (electrospray), the ability to size the fragments prior tomass spectrometric analysis is of great utility, particularly whentrying to perform more than one primer extension at a time (i.e.multiplexing). When a number of primers (and their respective extensionproducts) are to be analyzed by mass spectrometry and a target productrequiring determination is a 20-mer, it is theoretically possible tohave masses from 18-mers to 22-mers overlapping with the theoreticalmasses of the 20-mer product. Therefore, separating the primer extensionproducts on the basis of size prior to their analysis by massspectrometry effects a real-time deconvolution of the data, thus makinginterpretation more simple.

TABLE B Lowest mass Highest mass # mers (N) (poly-C_(N)) (poly-G_(N)) 185202 amu 5922 amu 19 5491 6251 20 5780 6580 21 6069 6909 22 6358 7238

There are instances where a probe is used to interrogate a site within atemplate (DNA or RNA), and this probing event is monitored by a numberof means. This is classically performed by sizing the fragments on agel, and then probing the resultant separation with a radioactive probe,“Southern” blotting for DNA, and “Northern” blotting for RNA.

There are other probes available which can be analyzed once they aredisplaced from the original template. As an example, some of theseprobes carry exogenous “mass tags” which serve to identify the probeonce the tag is cleaved off (often by photolysis).

However, to detect this “mass tag” in an efficient manner, the probe towhich it is associated must first be released from the template (See PCTapplications Nos. WO 9905322, WO 9905321, WO 9905320, WO 9905319, WO9727327, WO 9727325, WO 9905308, WO 9904896, WO 9727331, the entirecontents of all of the above being incorporated by reference). This canbe done by a means very similar to that presented above for releasingprimer extension products. The amplified products are denatured andprobed with the “mass tagged” probes. These duplexes are introduced tothe DNA Chromatography matrix under highly denaturing conditions(i.e. >75° C.), so that the probes and corresponding template areseparate from one another and retained at the top of the DNAChromatography matrix. Under suitable sizing conditions for ssDNA, theprobes are then selectively eluted from the DNA Chromatography matrix.In this case, instead of conducting the probes directly to a massspectrometer (as in the case of primer extension products), the “masstags” are cleaved from the probes and then conducted to the massspectrometer for detection (and hence detection of the probing event).This can further simplify the detection of “mass tags” by providinganother dimension along which selectivity can be provided, i.e., thedimension of probe length.

This invention is further illustrated by the following specific butnon-limiting examples where the descriptions of methods in the pasttense represent completed laboratory experiments. Descriptions in thepresent tense have not been carried out in the laboratory and are hereinconstructively reduced to practice by the filing of this application.

EXAMPLE 1 Size Fractionization of DNA Restriction Fragments with MIPCDNA Fragment Analysis System

One application of the WAVE® System is accurate and rapid sizing of DNAfragments. This feature can be exploited for the isolation of DNAfragments. Here we describe the method and buffer gradients used forsize fractionation and isolation of restriction fragments generated bydigestion of pUC 18 with Haelli. The nine fragments isolated range insize from 80 to 587 base pairs.

Liquid chromatography (LC) is a powerful technique for the separation ofnucleic acids due to its high resolving capability, short analysis time,and ease of recovery of DNA fragments for subsequent studies. The WAVE®System combines the precision of ion-pair reversed-phase LC withautomated sampling, data acquisition and reporting functions. Fragmentscan easily be collected and used in downstream experiments such assubcloning, amplification and sequencing.

TABLE C Protocol for DNA fragment sizing based on pUC 18/Haelll digestanalysis at 50° C. Buffer B Buffer A 0.1M TEAA, Flow Rate Time [min]0.1M TEAA 25% ACN mL/min 0.0 65 35 0.75 3.0 45 55 10.0 35 65 13.0 35 6514.0 0 100 15.5 0 100 16.5 65 35

Table D shows fragment sizing of pUC 18 plasmid cut with Haell using theprotocol in Table C on a 4.6×50 mm column containingoctadecylalkyl-substituted styrene-divinylbenzene copolymer beads(DNASep® column, Transgenomic, Inc., Omaha, Nebr.). A total of 9.96 μgof digested DNA was injected in a 20 μl volume. Table D shows amounts ofDNA per fragment based on a 9.96 μg injection. Fractions were collectedmanually.

TABLE D Amounts of DNA per restriction fragment based on a 9.96 μg totalinjection. Fragment Length Percentage of Relative Amount DNA base pairsPlasmid Length μg  80 3.01 0.30 102 3.84 0.38 174 6.55 0.65 257 9.670.96 267 10.05 1.00 298 11.22 1.12 434 16.33 1.63 458 17.24 1.72 58722.09 2.20

Peaks corresponding to the 80, 102,174, 298 and 587 bp fragments werecollected in 200 μl volumes. The 257/267 fragments, which differ only by10 bp, elute within less than 20 seconds of each other so do the 434/458bp fragments. In order to achieve separation of these peaks the fractionsize was lowered to 100 μl. After collection fractions were dried downand resuspended in 30 μl TE buffer (10 mM Tris, 1 mM EDTA) of which 25μl were re-injected for analysis using the protocol in Table C. Fragmentsizes and corresponding retention times are listed in Table E.

TABLE E Restriction fragment retention time comparison between aninjection of complete digest and purified individual restrictionfragments. Retention Time Retention Time Fragment Length, min, min, basepairs Complete Digest Individual Fragments  80 4.75 4.76 102 5.30 5.32174 6.71 6.77 257 8.54 8.69 267 8.81 8.92 298 9.46 9.60 434 11.48 11.69458 11.80 12.01 587 12.89 13.11

The results obtained are shown in FIGS. 12 to 21 wherein the figurenumber and the base pair length are shown in Table F.

TABLE F Fragment Length, base pairs FIG. No.  80 13 102 14 174 15 257 16267 17 298 18 434 19 458 20 587 21

DNA fragments ranging in size from 80 to 587 bp were separated andrecovered as shown in FIGS. 12-21. The 80, 102, 174, 257, 298, 434, and587 bp DNA fragments were fully purified, without contamination by anyother fragments of the restriction digest. Only the isolation of the 267bp fragment and 458 bp fragment, which are preceded by fragments in thedigest of 257 and 434 bp , respectively, showed minor contamination withthe corresponding shorter fragment.

EXAMPLE 2 Preparation of Spin Column

A 50 mg portion of resin is added to each spin column (See schematicrepresentation of column in FIG. 7) while pulling a vacuum on thecolumns. The sides of the columns were tapped to remove resin from thewalls. A polyethylene filter was placed on the top of the resin in eachvial, followed by a retaining ring, with gentle tapping with a hammer toposition the retaining ring securely against the filter. The spincolumns were washed with an aqueous solution containing 50% acetonitrile(ACN) and 0.1 M triethylammonium acetate (TEAA). The vials were thenwashed with an aqueous solution of 25% ACN and 0.1 M TEAA and then withan aqueous solution of 0.1 M TEAA.

EXAMPLE 3 Separation of PUC 18 Msp I with Spin Column

A sample solution was prepared by diluting 35 ml stock pUC 18 Msp I to 1ml with 0.1 M TEAA (1 ml total volume). A 400 ml aliquot (correspondingto 6.6 mg loaded on the column) was selected. Base pair lengthseparation of the solution was performed using the WAVE separationsystem (Transgenomic, Inc., Omaha, Nebr.) described in FIGS. 1-3, andthe chromatograms obtained for two of the columns are shown in FIG. 22for the pUC 18 Msp I standard.

An aliquot of the sample solution was pipetted into separate spincolumns and left standing for 5 min. Each vial was centrifuged at 5000rpm for 5 min. Then 400 μl of freshly prepared aqueous solutioncontaining 9.5% ACN and 0.1 M TEAA was pipetted into each spin column,each vial was left standing for 5 min, each vial was centrifuged at 5000rpm for 5 min, and the filtrate was analyzed using the WAVE separationsystem. The chromatogram obtained for the eluant is shown in FIG. 23 forthe pUC 18 Msp I standard. The treatment procedure was repeated.

The above procedure was repeated, replacing the 38% B solution with 100μl of a 100% B solution. The chromatogram by analyzing. the eluant withthe WAVE separation system is shown in FIG. 24 for the pUC 18 Msp Istandard.

This example demonstrates the removal of smaller size fragments from thecolumn while retaining the larger-sized fragments on the column, andsubsequent removal of the larger-sized fragments from the column. Thisis particularly useful for purifying a larger-sized fragment orfragments from smaller size contaminants.

EXAMPLE 4 Separation of pBR322HAE III with Spin Column

A sample solution was prepared by diluting 18 ml stock pbr322 HAE IIIdigest to 1 ml with 0.1 M TEAA (1 ml total volume). A 400 ml aliquot(corresponding to 6.6 mg loaded on column) was selected. Base pairlength separation of the solution was performed using the WAVEseparation system (Transgenomic, Inc., Omaha, Nebr.) described in FIGS.1-3, and the chromatograms obtained for two of the columns are shown inFIG. 25 for the pBR322HAE III standard.

An aliquot of the sample solution was pipetted into separate spincolumns and left standing for 5 min. Each vial was centrifuged at 5000rpm for 5 min. Then 400 μl of freshly prepared aqueous solutioncontaining 38% B (B is an aqueous 25% ACN solution, 0.1 M TEAA) waspipetted into each spin column, each vial was left standing for 5 min,each vial was centrifuged at 5000 rpm for 5 min, and the filtrate wasanalyzed using the WAVE separation system. The chromatogram obtained forthe eluant is shown in FIG. 26 for the pBR322HAE III standard. Thetreatment procedure was repeated.

The above procedure was repeated, replacing the 38% B solution with 100μl of a 100% B solution. The chromatogram by analysis with the eluantwith the WAVE separation system is shown in FIG. 27 for the pBR322HAEIII standard.

This example demonstrates the removal of smaller size fragments from thecolumn while retaining the larger-sized fragments on the column, andsubsequent removal of the larger-sized fragments from the column. Thisis particularly useful for purifying a larger-sized fragment orfragments from smaller size contaminants.

EXAMPLE 5 Purification of PCR Product with Spin Column

A sample solution was prepared by pipetting 100 μl 0.2 M TEAA onto thespin column and pipetting 100 μl of a 200 bp fragment (p53 exon 6genomic DNA) which had been amplified by PCR. Separation of the solutionwas performed using the WAVE separation system (Transgenomic, Inc.,Omaha, Nebr.) described in FIGS. 1-3, and shown in FIG. 28.

An aliquot of the sample solution was pipetted into separate spincolumns and left standing for 2 min. Each vial was centrifuged at 5000rpm for 5 min. Then 400 μl of freshly prepared aqueous solutioncontaining 38% B (B is an aqueous 25% ACN solution, 0.1 M TEAA) waspipetted into each spin column, each vial was left standing for 2 min,each vial was centrifuged at 5000 rpm for 5 min, and the filtrate wasanalyzed using the WAVE separation system. The treatment procedure wasrepeated.

The above procedure was repeated, replacing the 38% B solution with 100μl of a 100% B solution. The chromatogram by analyzing the eluant withthe WAVE separation system is shown in FIG. 29 for the 200 bp fragment.FIG. 29 shows a purified PCR product recovery of 97.9% and a byproductremoval of greater than 99.2%

This example demonstrates the ability of this procedure to elute PCRproduct with a high recovery and almost complete removal of PCRbyproducts.

EXAMPLE 6 Separation of a Labeled Oligonucleotide of the same Length byMIPC

A 200 bp fragment (from plitmus) using one biotinylated primer wasinjected onto an MIPC column and was eluted under gradient conditions toproduce a chromatogram as shown in FIG. 30. Separation was performedusing the WAVE separation system (Transgenomic, Inc., Omaha, Nebr.)described in FIGS. 1-3. The mobile phase comprised component A, 0.1Mtriethylammonium acetate (TEAA) and component B, 0.1M TEAA, 25%acetonitrile. A linear gradient was used in the separation starting at30% component B and extended to 70% component B in 12 minutes at 75° C.The column was a DNASep™ (Transgenomic, Inc. San Jose, Calif.).

In FIG. 30, the biotinylated oligonucleotide is eluted later than thenon-biotinylated oligonucleotide. The higher retention time is due tothe hydrophobicity of the biotin moeity. Thus, under denaturingconditions, the 200 nucleotide ssDNA species can be separated from thebiotinylated ssDNA species of the same length.

EXAMPLE 7 Kinetic Separation of Homoduplexes and Heteroduplexes by MIPC

A 209 bp fragment from human Y chromosome, locus DYS271 with an A to Gmutation at position 168 is chromatographed by MIPC and the result issubstantially similar to that seen in FIG. 31.

MIPC analysis conditions using the WAVE® DNA Fragment Analysis System,are as follows: Component A: 0.1M TEAA, Component B: 0.1M TEAA, 25%Acetonitrile; flow rate: 0.9 mL/min; temperature: 56° C.; detection: UV254 nM. The gradient used is as follows:

TIME, min. % A % B 0.0 46 54 0.5 45 55 4.0 38 62 4.1 0 100 4.5 0 100 4.646 54 5.0 46 54

The heteroduplexes would elute before the homoduplexes.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

The invention claimed is:
 1. An ambient or low pressure device forseparating polynucleotide fragments from a mixture of polynucleotidefragments comprising: a tube having an upper solution input chamber, alower eluant receiving chamber, and a fixed unit of Matched IonPolynucleotide Chromatography separation media supported therein, theseparation media having nonpolar separation surfaces which are free frommultivalent cations which would react with counterion to form aninsoluble polar coating on the surface of the separation media.
 2. Anambient or low pressure device of claim 1 wherein the separation mediais a member selected from the group consisting of beads, capillarychannels and monolith structure.
 3. An ambient or low pressure device ofclaim 2 wherein the fixed unit of separation media comprise a fixed bedof separation media particles.
 4. An ambient or low pressure device ofclaim 3 wherein the separation media particles are selected from thegroup consisting of organic polymer and inorganic particles having anonpolar surface.
 5. An ambient or low pressure device of claim 1wherein the lower chamber is closed.
 6. An ambient or low pressuredevice of claim 1 wherein the lower chamber has an open bottom portion.7. An ambient or low pressure device of claim 6 in combination with aeluant container shaped to receive said lower chamber.
 8. An ambient orlow pressure device of claim 7 wherein the eluant chamber is acentrifuge vial.
 9. An ambient or low pressure device of claim 7 whereinthe cylinder is a member of an array of cylinders and the eluantcontainer is a member of an array of eluant containers, and the array ofcylinders and array of containers have matching configurations.
 10. Anambient or lower pressure separation system comprising a combination ofmulticavity separation plate having outer sealing edges, a multiwellcollection plate and a vacuum system having a separation plate sealingmeans forming a sealed engagement with the outer sealing edges of themulticavity separation plate and a vacuum cavity receiving the multiwellcollection plate; the multicavity separation plate including an array oftubes, each tube having an upper solution input chamber, a lower eluantreceiving chamber with an bottom opening therein, and a fixed unit ofMatched Ion Polynucleotide Chromatography separation media supportedtherein, the separation media having nonpolar separation surfaces whichare free from multivalent cations which would react with counterion toform an insoluble polar coating on the surface of the separation media;the multiwell collection plate having collection wells which arepositioned to receive liquid from the bottom opening of the lower eluantreceiving chamber.
 11. An ambient or lower pressure separation system ofclaim 10 wherein the separation media is a member selected from thegroup consisting of beads, capillary channels and monolith structures.12. An ambient or low pressure device of claim 11 wherein the fixed unitof separation media comprise a fixed bed of separation media particles.13. An ambient or low pressure device of claim 12 wherein the separationmedia particles are selected from the group consisting of organicpolymer and inorganic particles having a nonpolar surface.